Association of proteins
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Association of proteins with the surface of intracellular membranes is essential for a wide variety of cellular functions — from signalling and trafficking to main- taining cell structure — and involves an ever-growing array of lipid-binding domains. For example, membrane anchoring (and dynamics) of the cytoskeleton requires the direct interaction of lipid-binding domains with the membrane surface 1,2 . Moreover, upon stimulation of cell surface receptors, numerous signalling proteins are transiently recruited to specific locations in plasma (and other) membranes, where they exert their func- tions (such as lipid modification or activation of small GTPases) or become effectively co-localized with part- ners in a signal-transduction pathway 2,3 . Some cellular compartments are ‘marked’ by the presence of specific lipids, and recognition of these lipids is required for the intracellular trafficking machinery to discern one intracellular organelle from another 4 . Despite common themes, including the almost exclusive use of acidic phospholipids as binding targets, the domains and proteins that bind membrane surfaces vary widely in their binding mechanisms, which allows differences to be exploited for distinct modes of control. In this review, I discuss how different lipid-binding domains associate with membranes, illustrating where possible both the similarities and distinctions between domains and how their individual properties are ideally suited for their particular cellular function. To be able to include some detail, I focus only on domains that have well defined globular structures and on those that bind their target lipids in a membrane context. Readers are referred to excellent recent reviews 5,6 for a discussion of membrane association by unstructured clusters of basic and hydrophobic residues, which share some functions with the domains that are discussed here. Targets of phospholipid-binding domains The main acidic phospholipids in mammalian cell mem- branes are phosphatidylserine, phosphatidic acid and phosphatidylinositol. In addition, a small proportion of the membrane phosphatidylinositol is phosphorylated at the 3-, 4- and/or 5-positions to generate phospho- inositides. The approximate ratios of the different acidic phospholipids in mammalian cells are listed in BOX 1, although precise levels vary considerably depending on the cell type and growth conditions. The phospho- inositides are always very minor species; indeed, in the inner leaflet of the plasma membrane of normal cells, phosphatidylinositol-(4,5)-bisphosphate (PtdIns(4,5)P 2 ) is estimated to account for only 0.5–1.0% of phospholipid molecules, whereas phosphatidylserine accounts for ~25–35% 5 . However, phosphoinositides have a dispro- portionately major role in directing the membrane asso- ciation of phospholipid-binding domains, as discussed below, and their levels are acutely regulated 2,7 . Diverse phospholipid-binding domains At least 10 different globular domain types bind phospho- lipids at the membrane surface (TABLE 1). The interactions of these domains with the membrane surface fall into two broad classes: some are highly specific and involve stereospecific recognition of particular membrane components; others are non-specific and involve attraction to a general physical property of the membrane (such as charge, amphiphilicity and curvature). These two extremes are spanned by the domains listed in TABLE 1. Indeed, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 809C Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104-6059, USA. e-mail: [email protected]. upenn.edu doi:10.1038/nrm2328 Inner leaflet A lipid layer that faces the inside of the cell. Stereospecificity Specific recognition of a particular stereoisomer in a binding reaction. Amphiphilicity Possession of both hydrophobic and hydrophilic regions. Membrane recognition by phospholipid-binding domains Mark A. Lemmon Abstract | Many different globular domains bind to the surfaces of cellular membranes, or to specific phospholipid components in these membranes, and this binding is often tightly regulated. Examples include pleckstrin homology and C2 domains, which are among the largest domain families in the human proteome. Crystal structures, binding studies and analyses of subcellular localization have provided much insight into how members of this diverse group of domains bind to membranes, what features they recognize and how binding is controlled. A full appreciation of these processes is crucial for understanding how protein localization and membrane topography and trafficking are regulated in cells. NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 9 | FEBRUARY 2008 | 99 REVIEWS FOCUS ON LIPIDS © 2008 Nature Publishing Group
Transcript of Association of proteins
Association of proteins with the surface of intracellular membranes is essential for a wide variety of cellular functions — from signalling and trafficking to main-taining cell structure — and involves an ever-growing array of lipid-binding domains. For example, membrane anchoring (and dynamics) of the cytoskeleton requires the direct interaction of lipid-binding domains with the membrane surface1,2. Moreover, upon stimulation of cell surface receptors, numerous signalling proteins are transiently recruited to specific locations in plasma (and other) membranes, where they exert their func-tions (such as lipid modification or activation of small GTPases) or become effectively co-localized with part-ners in a signal-transduction pathway2,3. Some cellular compartments are ‘marked’ by the presence of specific lipids, and recognition of these lipids is required for the intracellular trafficking machinery to discern one intracellular organelle from another4.
Despite common themes, including the almost exclusive use of acidic phospholipids as binding targets, the domains and proteins that bind membrane surfaces vary widely in their binding mechanisms, which allows differences to be exploited for distinct modes of control. In this review, I discuss how different lipid-binding domains associate with membranes, illustrating where possible both the similarities and distinctions between domains and how their individual properties are ideally suited for their particular cellular function. To be able to include some detail, I focus only on domains that have well defined globular structures and on those that bind their target lipids in a membrane context. Readers are referred to excellent recent reviews5,6 for a discussion of membrane association by unstructured clusters of basic
and hydrophobic residues, which share some functions with the domains that are discussed here.
Targets of phospholipid-binding domainsThe main acidic phospholipids in mammalian cell mem-branes are phosphatidylserine, phosphatidic acid and phosphatidylinositol. In addition, a small proportion of the membrane phosphatidylinositol is phosphorylated at the 3-, 4- and/or 5-positions to generate phospho-inositides. The approximate ratios of the different acidic phospholipids in mammalian cells are listed in BOX 1, although precise levels vary considerably depending on the cell type and growth conditions. The phospho-inositides are always very minor species; indeed, in the inner leaflet of the plasma membrane of normal cells, phosphatidylinositol-(4,5)-bisphosphate (PtdIns(4,5)P2) is estimated to account for only 0.5–1.0% of phospholipid molecules, whereas phosphatidylserine accounts for ~25–35%5. However, phosphoinositides have a dispro-portionately major role in directing the membrane asso-ciation of phospholipid-binding domains, as discussed below, and their levels are acutely regulated2,7.
Diverse phospholipid-binding domainsAt least 10 different globular domain types bind phospho-lipids at the membrane surface (TABLE 1). The interactions of these domains with the membrane surface fall into two broad classes: some are highly specific and involve stereospecific recognition of particular membrane components; others are non-specific and involve attraction to a general physical property of the membrane (such as charge, amphiphilicity and curvature). These two extremes are spanned by the domains listed in TABLE 1. Indeed,
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 809C Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104-6059, USA. e-mail: [email protected]:10.1038/nrm2328
Inner leafletA lipid layer that faces the inside of the cell.
StereospecificitySpecific recognition of a particular stereoisomer in a binding reaction.
AmphiphilicityPossession of both hydrophobic and hydrophilic regions.
Membrane recognition by phospholipid-binding domainsMark A. Lemmon
Abstract | Many different globular domains bind to the surfaces of cellular membranes, or to specific phospholipid components in these membranes, and this binding is often tightly regulated. Examples include pleckstrin homology and C2 domains, which are among the largest domain families in the human proteome. Crystal structures, binding studies and analyses of subcellular localization have provided much insight into how members of this diverse group of domains bind to membranes, what features they recognize and how binding is controlled. A full appreciation of these processes is crucial for understanding how protein localization and membrane topography and trafficking are regulated in cells.
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Second messengersMolecules that act in a cell to promote responses to extracellular stimuli.
Phorbol estersPolycyclic esters that are isolated from croton oil. The most common are phorbol‑12‑myristate‑13‑acetate and 12‑O‑tetradecanoyl‑phorbol‑ 13‑acetate. These are both potent carcinogens or tumour promoters because they mimic diacylglycerol and thereby irreversibly activate protein kinase C.
they are even spanned within certain domain classes. The degree (and type) of specificity in phospholipid binding has important functional implications. naturally, a domain that recognizes only the most general proper-ties of a membrane could associate with all intracellular membranes all of the time. Some temporal (but not neces-sarily spatial) specificity can be introduced if membrane association requires the presence of a soluble second messenger such as Ca2+. Some spatial (and temporal) specificity can also be introduced if the phospholipid-binding domain has a preference for membrane surfaces with high curvature. both of these mechanisms are used in biology, but the most specific cases involve domains that selectively recognize rare membrane components that are restricted in their location, time of synthesis or both. Among phospholipids, the phosphoinositides represent the best examples of acutely regulated binding targets (BOX 1), and all phosphoinositides except PtdIns4P and PtdIns5P have one or more well characterized specific binding domains.
Target-specific binding domainsThe first domains that were shown to recognize mem-branes in a target-specific manner were C1 (defined below)8 and pleckstrin homology (PH)9 domains. C1 domains are considered ‘honorary’ phospholipid-binding domains here, as their physiological binding target (diacylglycerol (DAG)) is one phosphate group short of qualifying as a phospholipid.
Fused to green fluorescent protein (GFP), the target-specific domains described in this section pro-vide a set of convenient ‘probes’ for monitoring intra-cellular localization (FIG. 1) and levels of their specific targets10,11.
C1 domains. C1 domains are named after ‘conserved region-1’ from protein kinase C (PKC), and were identified almost 20 years ago as the binding sites that are responsible for PKC activation by phorbol esters and DAG8. They are zinc‑finger domains of ~50 amino acids that contain the signature motif HX12CX2CX13–14 CX2CX4HX2CX7C (in which C is cysteine, H is histi-dine and X is any residue). Typical C1 domains all bind DAG and are found in PKC isoforms and DAG kinases8. Atypical C1 domains do not bind phorbol esters or DAG, and their function is not clear.
A crystal structure of the (typical) C1b domain from PKCδ (which contains two C1 domains: C1A and C1b) has provided important insights into how this domain associates with membranes12. A band of hydrophobic side chains encircles the DAG/phorbol ester-binding site and penetrates the apolar milieu of the membrane (FIG. 2). C1 domains bind 10–80 fold more strongly to phorbol esters that are embedded in phosphatidylserine membranes than to the same free phorbol esters13. This difference reflects the contribution of multiple driving forces to membrane association by C1 domains: that is, direct (1:1) interaction with the target ligand (DAG or phorbol ester), membrane partitioning of hydro-phobic side chains, plus electrostatic attraction of key basic residues in the C1 domain to phosphatidylserine headgroups. This combination of driving forces is required for membrane targeting in vivo, with the acute generation of DAG functioning as the trigger10.
Phosphoinositide-specific PH domains. The name ‘plecks-trin homology’ reflects the identification of a ~100-amino-acid region of sequence homology that occurs twice in pleckstrin (the major PKC substrate in plate-lets), and in numerous other proteins with membrane- associated functions9. The n-terminal PH domain from pleckstrin was found to bind phosphoinositides14, and studies of the PH domain from phospholipase Cδ1 (PlCδ1) provided the first evidence for stereospecific rec-ognition of a phosphoinositide headgroup by a protein domain15,16. The PlCδ1 PH domain (PlCδ–PH) binds strongly to either PtdIns(4,5)P2 or to its isolated head-group, d-myo-inositol-1,4,5-trisphosphate (Ins(1,4,5)P3). In contrast to the C1 domain, PlCδ–PH actually binds more strongly to isolated Ins(1,4,5)P3 than to the head-group of membrane-embedded PtdIns(4,5)P2. Moreover, Ins(1,4,5)P3 efficiently displaces PtdIns(4,5)P2 from the
Box 1 | Relative levels of acidic phospholipids in mammalian cells
The main acidic phospholipids in mammalian cells are phosphatidylserine, phosphatidic acid and phosphatidylinositol, which constitute approximately 8.5%, 1.5% and 1.0%, respectively, of total lipid (by weight) in erythrocytes105. The phosphoinositides are much less abundant, and their approximate relative levels have been estimated by Stephens et al.7 (see table). Estimated fold increases in the levels of each phosphoinositide in response to selected treatments are also shown. Phosphatidylinositol‑(3,4,5)‑trisphosphate (PtdIns(3,4,5)P3) and PtdIns(3,4)P2 levels are robustly (but transiently over 10–30 minutes) elevated when phosphoinositide 3‑kinases (PI3Ks) are activated by agonists for many cell‑surface receptors106. The levels of PtdIns(4,5)P2 fall slightly following its conversion by phospholipase‑C‑mediated hydrolysis (to generate inositol trisphosphate (Ins(1,4,5)P3) and diacylglycerol) and PI3K‑mediated phosphorylation. The fall in PtdIns4P levels primarily reflects its 5‑phosphorylation to replenish pools of PtdIns(4,5)P2. PtdIns5P levels were shown to be elevated in platelets following thrombin treatment107, in response to various stresses and during the cell cycle108. PtdIns3P levels seem to be relatively constant, although PtdIns3P is restricted primarily to endosomes and multivesicular bodies. Finally, PtdIns(3,5)P2 is implicated in several cellular processes58 and its levels are elevated in response to cellular stress. Note that PtdIns5P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 have not been detected in Saccharomyces cerevisiae. However, PtdIns(3,4,5)P3 (but not PtdIns(3,4)P2 or PtdIns5P) has been detected in Schizosaccharomyces pombe109.
lipid relative level (%)*‡ Fold increase on stimulation
Phosphatidylserine 8.5 1
Phosphatidic acid 1.5 1
Phosphatidylinositol 1.0 1
PtdIns3P 0.002 1§
PtdIns4P 0.05 0.7||
PtdIns5P 0.002 3–20¶
PtdIns(4,5)P2 0.05 0.7||
PtdIns(3,4)P2 0.0001 10||
PtdIns(3,5)P2 0.0001 2–30#
*Phosphoinositide values are taken from Stephens et al.7. ‡Relative levels of total phosphatidylserine, phosphatidylinositol and phosphatidic acid reflect human erythrocyte values from Tanford105. §Note that acute insulin‑induced PtdIns3P production has also been reported110. ||Estimated changes upon ligand stimulation of neutrophils106. ¶In response to thrombin stimulation, cellular stress and during the cell cycle108. PtdIns5P has not been described in yeasts111. #PtdIns(3,5)P2 levels increase by up to 30‑fold in yeast and 2–6‑fold in plant and animal cells in response to hyperosmotic stress58.
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Zinc fingerA small structural motif that is found in many proteins, including phospholipid‑binding proteins, DNA‑binding proteins and ubiquitin ligases. Zinc fingers are characterized by particular sequences of cysteines and histidines that coordinate bound Zn2+ ions. The bound Zn2+ ions are structurally crucial, and their ability to nucleate the protein structure obviates the need for a hydrophobic core.
Guanine nucleotide-exchange factorA protein that facilitates the exchange of GDP for GTP in the nucleotide‑binding pocket of a GTP‑binding protein.
AgammaglobulinaemiaA disorder that is caused by an inability to make mature B cells and, as a result, antibodies. X‑linked agammaglobulinaemia can arise from mutations in the PH domain of Bruton’s tyrosine kinase (BTK) that block the ability of BTK to respond to phosphoinositide 3‑kinase signalling. Activation of BTK is crucial for B‑cell maturation.
PlCδ–PH-binding site, which leads to the dissociation of the PH domain from membranes when Ins(1,4,5)P3 is generated in cells17 or added in vitro18.
A crystal structure19 of PlCδ–PH bound to Ins(1,4,5)P3 provided a clear view of how this PH domain recog-nizes the pattern of phosphate groups that is specific to PtdIns(4,5)P2. This structure also indicated that membrane binding by PlCδ–PH need not involve sig-nificant membrane penetration. There is no equivalent of the band of hydrophobic side chains that surrounds the C1 domain ligand-binding site, although PlCδ–PH does show membrane-insertion activity under some circumstances20.
PH domains are well known effectors of the lipid second messengers PtdIns(3,4,5)P3 and PtdIns(3,4)P2, which are generated transiently upon activation of almost all cell surface receptors21. A small subclass of PH domains, including those from bruton’s tyrosine kinase (bTK)22,23, general receptor for phosphoinositides-1 (GRP1)24 and protein kinase b (PKb; also known as AKT)25, recognize one or both of these second messen-gers with remarkable specificity and affinity. These PH domains represent classic examples of signal-regulated membrane-targeting modules. In each case, the isolated PH domain (as a GFP fusion protein) is predominantly cytosolic in unstimulated cells, but undergoes a dramatic transient relocalization to the plasma membrane on sig-nal-dependent activation of phosphoinositide 3-kinase (PI3K)11,26. The selectivity and affinity characteristics of these PH domains allow them to target only membranes that contain PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2, despite PtdIns(4,5)P2 being present at 10–20-fold higher levels (BOX 1). This highly specific recognition of PI3K products is responsible for signal-dependent membrane recruitment (and activation) of kinases such as bTK and PKb/AKT,
guanine nucleotide‑exchange factors (GeFs) such as GRP1, and other key signalling molecules21. Amino-acid subs-titutions in PH domains that abolish PtdIns(3,4,5)P3 binding cause severe signalling defects, as seen in X-linked agammaglobulinaemia when the bTK PH domain is mutated27. Conversely, mutations that promote consti-tutive association of certain PH domains with the plasma membrane can cause cancer, as was recently reported for PKb/AKT28. Intriguing studies29 have also indicated that certain PtdIns(3,4,5)P3-specific PH domains might be positively regulated by the soluble headgroup inositol- (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4), although mechanistic details remain unclear.
Crystal structures have provided insight into phos-phoinositide recognition by PH domains30. All high- affinity, stereospecific PH domains share a similar phosphoinositide-binding site. PH domains have a 7-stranded β-sandwich structure, shown in FIG. 2, and the β1–β2 loop between the first two β-strands functions as a ‘platform’ for the interaction with the phosphoinositide headgroup. This loop lines a deep binding pocket and contains the sequence motif31 KXn(K/R)XR, in which the basic side chains (from K (lysine) and R (arginine)) form most phosphate-group interactions. basic (and other) side chains from elsewhere in the domain make additional contacts that define the preferred inositol ring orientation and phosphorylation pattern. Distinct phosphoinositide-binding specificities are largely attributable to variations on the same theme, and robust rules have been defined for predicting ligand preference on the basis of sequence30,32.
In addition to this ‘canonical’ mode of phospho-inositide recognition by PH domains, several PH domains, including those from the GeF TIAM1 and from the Rho GTPase‑activating protein-9 (ARHGAP9), achieve modestly specific and high-affinity phosphoinositide
Table 1 | Occurrence of selected phospholipid-binding domains in commonly studied organisms
Domain* Humans Mus musculus Caenorhabditis elegans
Drosophila melanogaster
Saccharomyces cerevisiae
PH 303 (258) 284 (241) 79 (71) 81 (73) 32 (29)
PKC C2‡ 200 (125) 200 (126) 70 (48) 62 (43) 22 (11)
C1 79 (58) 77 (57) 48 (34) 38 (28) 2 (1)
PX 35 (35) 39 (39) 11 (11) 14 (14) 15 (15)
FYVE 27 (26) 28 (27) 17 (17) 12 (12) 6 (5)
Discoidin C2ठ24 (18) 24 (18) 3 (3) 7 (5) -
GRAM 18 (15) 19 (15) 5 (4) 5 (4) 7 (6)
F-BAR|| 14 (14) 17 (17) 3 (3) 4 (4) 4 (4)
Annexin 56 (13) 51 (12) 15 (4) 18 (6) -
Gla§ 13 (13) 17 (17) - - -
N-BAR 9 (9) 12 (12) 5 (5) 3 (3) 2 (2)
ENTH/ANTH 9 (9) 7 (7) 5 (5) 3 (3) 8 (8)The number of examples of each domain (with the number of proteins represented in parentheses) was obtained using the SMART database116 (see Further information) in Genomic Mode in July 2007. The number of examples of each domain quoted here is directly comparable with those quoted for various modular signalling domains by Bhattacharyya et al.117. *It should be noted that these domains are identified solely by sequence homology. Their function as phospholipid-binding domains may not be fully conserved across (or within) species. ‡Note that PKC-class and discoidin-class C2 domains are not related. §Discoidin C2 domains and Gla domains are the only extracellular phospholipid-binding domains listed. All others are intracellular. ||Termed FCH domain in the SMART database. ANTH, AP180 N-terminal homology; BAR, Bin, amphiphysin and Rvs; C1, conserved region-1; C2, conserved region-2; ENTH, epsin N-terminal homology; FYVE, Fab1, YOTB, Vac1, EEA1; Gla, γ-carboxyglutamate rich; GRAM, glucosyltransferases, Rab-like GTPase activators and myotubularins; PH, pleckstrin homology; PKC, protein kinase C; PX, Phox homology.
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O
OH
O O
DAG
O
O
O O
P
O O–
O
HO
HOOH
OHOH
OP
O O–
O
HO
HO OHOH
OP
O O–
O
HO
OHOH
OP
O O–
O
HO
OH
OP
O O–
O
HO
OHOH
OH
OP
O O–
O
HO
OH
OH
OP
O O–
O
HO
HOOH
OH
OP
O O–
O
HO
HOOH
PtdIns3P
PtdIns
PtdIns4P PtdIns(3,4)P2
PtdIns5P PtdIns(4,5)P2
PtdIns(3,4,5)P3
PtdIns(3,5)P2
PPP
P
P P
P
P
P P P
P
PtdIns3P 5-KPROPPINs
PH domains
PH domains
PHD fingers
C1 domains
PX domains FYVE domains
PtdIns 4-K
?
PtdIns5P 4-K
PI3K
SHIPs
PtdIns(3,4)P2 5-K
PtdIns3P 4-K
PtdIns4P 5-K
PtdIns
3-K
PtdIns 5-K
PI3K
PTEN
12
3 45
6
GTPase-activating proteins(GAPs). Proteins that stimulate the intrinsic ability of a GTPase to hydrolyse GTP to GDP. GAPs negatively regulate GTPases by converting them from active states (GTP bound) to inactive states (GDP bound).
Split PH domainA pleckstrin homology (PH) domain with an interrupted sequence. Regions of polypeptide that are well separated in the primary sequence of a protein can interact with one another to form a globular PH domain fold. The interruptions are usually in the flexible loops of the PH domain and can harbour other domains.
binding through a distinct site33. This ‘non-canonical’ site is related to the binding site for Ins(1,4,5)P3 in the spectrin PH domain34 and lies on the opposite side of the β1–β2 loop from the canonical site (FIG. 2). Intriguingly, a similar site is thought to be responsible for the binding of PtdIns3P and other phosphoinositides to the ‘split PH domain’ that is found in the Glue (GRAM-like ubiquitin- binding in eap45) domain of vPS36 (a component of the endosomal sorting complex required for transport (ESCRT) II complex)35,36. These findings imply that at least one additional class of phosphoinositide-binding PH domain beyond that exemplified by those present in PlCδ1, bTK, GRP1 and PKb/AKT has yet to be fully characterized.
Genome-wide studies37 have shown that most Saccharomyces cerevisiae PH domains do not bind strongly or specifically to phosphoinositides. of the ~234 PH domains in the human proteome (TABLE 1), only ~10% are known to bind strongly and specifically
to phosphoinositides. Although several PH domains are well known (and well understood) as phosphoinositide-specific membrane-targeting domains, these constitute only a small minority of a large and poorly understood class of domains. The functions of the rest remain unclear, and other ligands are being sought38.
FYVE domains. All ‘Fab1, yoTb, vac1, eeA1’ (Fyve) domains specifically recognize PtdIns3P, which is pri-marily found in endosomes, multivesicular bodies and phagosomes39. like C1 domains, Fyve domains are zinc fingers. They contain 60–70 amino acids, com-prising two β-hairpins and a small C-terminal α-helix that are held together by two tetrahedrally coordinated Zn2+ ions40. A conserved basic motif (RR/KHHCR) in the first β-strand contributes to a shallow, positively charged binding pocket for PtdIns3P, and is responsible for all but two of the direct hydrogen bonds that exist between Fyve domains and PtdIns3P39,41.
Figure 1 | Domains that bind specific lipid targets. The structures and interconversion reactions are shown for all phosphoinositides that are found in mammalian cells. The phosphoinositide kinases that catalyse the addition of phosphate groups to the 3-, 4- and/or 5-positions are shown, as are the lipid phosphatases PTEN (phosphatase and tensin homologue on chromosome 10) and SHIP (SH2-containing inositol 5′-phosphatase). The phospholipid-binding domains that recognize specific phosphoinositides (and diacylglycerol (DAG)) are shown. Only β-propellers that bind phosphoinositides (PROPPINs) recognize phosphatidylinositol-(3,5)-bisphosphate (PtdIns(3,5)P2). Only pleckstrin homology (PH) domains recognize PtdIns(4,5)P2, PtdIns(3,4,5)P3 or PtdIns(3,4)P2 with high specificity. All ‘Fab1, YOTB, Vac1, EEA1’ (FYVE) domains bind PtdIns3P, as do nearly all Phox-homology (PX) domains (although there are a few mammalian exceptions that reportedly bind PtdIns(4,5)P2 or PtdIns(3,4)P2). Whether PtdIns4P-specific domains exist remains unclear37,104,112, although certain PH domains have been reported to prefer this lipid113. A split PH domain from VPS36 binds PtdIns3P36. The status of plant homeodomain (PHD) fingers as putative PtdIns5P effectors114 is not clear. PX, Phox homology.
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Membrane
Membrane
PKCδ C1
(EEA1 FYVE)2 ARHGAP9 PH p47phox PX
PLCδ1 PH p40phox PX
Zn2+
Zn2+
Zn2+
Zn2+
Hydrophobicband
Phorbolester
P4
P3P5
P4P3
P1 P1 P1
P5
P1Ins(1,4,5)P3
Ins(1,4,5)P3Ins(1,3)P2 Ins(1,3)P2
di-C4PtdIns3P
di-C4PtdIns3P(modelled)
Membraneinsertion
Membraneinsertion
Membraneinsertion
P3
P1
Proposedsecond anion-binding site
ESCRT(Endosomal sorting complex required for transport). The multiprotein ESCRT machinery (ESCRT‑I, ‑II and ‑III) promotes inward vesiculation at the limiting membrane of the sorting endosome and selects cargo proteins for delivery to the intralumenal vesicles of multivesicular bodies.
EndosomesVesicles that are formed by invagination of the plasma membrane.
Multivesicular bodiesEndosomal intermediates in which small membrane vesicles are enclosed in a limiting membrane. The internal vesicles are thought to form by invagination and budding from the limiting membrane.
PhagosomesMembrane‑bound vesicles that contain microorganisms or particulate material from the extracellular environment.
AvidityThe overall measure of binding between a multivalent ligand and its receptors, which reflects the combined strength of multiple binding sites. Avidity was originally defined for antibodies, for which it refers to the overall strength of binding between multivalent antigens and antibodies.
Coiled-coil domainA protein structural domain that often mediates subunit oligomerization. Coiled coils contain between two and five α‑helices that twist around each other to form a supercoil.
Sorting nexinAlso known as SNX proteins. These proteins are characterized by the presence of Phox‑homology (PX) domains and play roles in endosomal cargo sorting as well as other functions.
Fyve domains bind much more strongly to mem-brane-embedded PtdIns3P than to the isolated head-group (Ins(1,3)P2) or short-chain phosphoinositide39,41, and in this sense are more similar to C1 domains than to PH domains of the PlCδ–PH class. like C1 domains, Fyve domains gain additional binding energy from both membrane insertion and delocalized electrostatic attraction39,42,43. However, even the cooperation of these modes of interaction does not seem to be sufficient for in vivo targeting of some Fyve domains to PtdIns3P-containing endosomal membranes44. endosomal tar-geting of most Fyve domains is inefficient unless the Fyve domain is also dimerized to allow multivalent (increased avidity) binding to multiple PtdIns3P mole-cules in the same membrane44,45. As shown in FIG. 2, the Fyve domain from early endosome antigen-1 (eeA1) is preceded by a coiled‑coil domain that drives its dimer-ization and bivalent binding to PtdIns3P-containing membranes41.
PX domains. A region of 130 amino acids of sequence hom-ology that is found in components of the phagocyte nADPH oxidase (phox) complex, termed the Phox-homology or PX domain46, was identified as a PtdIns3P-binding domain47 in 2001. Most PX domains are found in sorting nexin (SnX) proteins48, which are important in membrane trafficking. Although all S. cerevisiae PX domains bind selec-tively to PtdIns3P, only 4 (of 15) bind with high affinity49. In mammals, there are also examples of PX domains that prefer PtdIns(3,4)P2 or PtdIns(4,5)P2 (REFS 50,51), but selectivity is not strong in these cases and the preferred ligand for most PX domains appears to be PtdIns3P48.
Structural studies52 indicate that the PX domains (FIG. 2) employ the membrane-association mechanism that is used by C1 and Fyve domains — with combined headgroup binding, electrostatic attraction and mem-brane insertion — rather than relying primarily on head-group interactions as PH domains do. Again, cooperation of several driving forces is required for high-affinity
Figure 2 | Structures of target-specific phospholipid-binding domains. The protein kinase Cδ (PKCδ) C1 domain (Protein Data Bank (PDB) code 1PTR) was solved as a complex with phorbol-1,3-acetate12. The two Zn2+ ions are labelled, as is the ‘hydrophobic band’ of residues that is thought to penetrate the membrane surface. The likely position of the membrane is approximated by the shaded bar. Two pleckstrin homology (PH) domains are shown. One is the phospholipase Cδ1 (PLCδ1) PH domain (PDB code 1MAI), which is bound to inositol-(1,4,5)-trisphosphate (Ins(1,4,5)P3) through the ‘canonical’ binding site19. The other is the ARHGAP9 PH domain (PDB code 2P0D), which binds Ins(1,4,5)P3 through the spectrin-like ‘non-canonical’ site33. Two Phox-homology (PX) domains are also shown. One is from p40phox, with bound dibutanoylphosphatidylinositol 3-phosphate (PtdIns3P) (PDB code 1H6H), which shows the phosphoinositide-binding site and membrane insertion loop52. The other is from p47phox (PDB code 1O7K), and shows PtdIns3P modelled into the phosphoinositide-binding site, plus a sulphate ion bound at the putative second anion-binding site, which is proposed to interact with phosphatidic acid64. A truncated ‘Fab1, YOTB, Vac1, EEA1’ (FYVE) domain dimer from early endosome antigen-1 (EEA1)41 bound to Ins(1,3)P2 is also shown (PDB code 1JOC), illustrating headgroup binding, membrane insertion and dimerization. Structurally crucial Zn2+ ions are marked. Yellow side chains are those that are likely to penetrate the membrane.
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RetromerA complex of five proteins (Vps35, Vps26, Vps29, Vps17 and Vps5 in yeast) that is important for recycling transmembrane proteins from endosomes to the trans‑Golgi network.
membrane targeting, with the presence or absence of PtdIns3P (or other cognate ligand) determining whether the overall binding energy is (or is not) sufficient to drive the domain to the membrane. A few isolated PX domains, such as those from p40phox and SnX3 (REFS 49,50,53,54), bind PtdIns3P-containing membranes with high affinity and can be recruited independently to target membranes. However, most PX domains bind weakly to PtdIns3P- containing membranes and are only targeted effectively to membranes when they are part of a multivalent complex. For example, SnX1 requires dimerization for its target-ing to endosomes55, and the yeast SnX proteins vps5 and vps17 are only recruited to PtdIns3P-containing mem-branes (through their low-affinity PX domains) as part of the oligomeric retromer56 complex, which is capable of multivalent interactions57.
PROPPINs. A search for potential effectors of PtdIns(3,5)P2 (REF. 58) in S. cerevisiae led to the identification of Atg18 as a new member of the family of phosphoinositide-binding proteins59. Atg18 is a 500-amino-acid β-propeller protein that binds PtdIns(3,5)P2 with high affinity and specificity59, and is the prototype for the PRoPPIns (β-propellers that bind phosphoinositides58) of which three are found in S. cerevisiae and four in humans.
In common with Fyve and PX domains, Atg18 seems to bind much more strongly to the membrane-embedded phosphoinositide than to its isolated headgroup or short-chain analogues (K. narayan and M.A.l., unpublished observations), which implies that membrane insertion and/or delocalized electrostatic attraction to the mem-brane surface is important in driving membrane associa-tion. Studies of other PRoPPIns, including those from Drosophila melanogaster (in which there are three) and humans, indicate that some bind both PtdIns(3,5)P2 and PtdIns3P58,60,61. no structure has yet been reported for a PRoPPIn–phosphoinositide complex.
Phosphatidic acid binding. Several proteins have been reported to recognize phosphatidic acid, including pro-tein kinases and phosphatases, cAMP-specific phospho-diesterases, transcription factors and others62. It has also been reported that both the son-of-sevenless (SoS) PH domain63 and the p47phox PX domain64 have a second anion-binding pocket, in addition to their phospho-inositide-binding sites, to which phosphatidic acid is thought to bind (FIG. 2). other than these examples, there is no clearly defined globular domain to which phos-phatidic acid-binding activity can be ascribed. Rather, the regions implicated in such binding tend to comprise
Table 2 | Phospholipid-binding domains at a glance
Domain Typical size (amino acids)
Structure Preferred target* membrane insertion?
ca2+ required?
Dimerization required?
refs
C1 ~50 Zn2+ finger DAG, phorbol esters Yes No No 8
PKC C2‡ ~130 β-sandwich PtdSer (and others) Yes Yes No 66
PH ~125 β-sandwich Phosphoinositides, quite diverse, some highly specific
Some reported20 No Some examples 9
FYVE 60–70 Zn2+ finger PtdIns3P Yes No Most cases 39
PX ~130 α+β structure PtdIns3P (a few bind other phosphoinositides)
Yes No Most cases 48
PROPPIN ~500 β-propeller PtdIns(3,5)P2 (PtdIns3P also in some cases)
Unknown No No 58
Gla ~45 α-helical (requires Ca2+ to fold)
PtdSer Yes Yes No 62
Annexin ~310 α-helical array Acidic phospholipids Unknown Yes No 71
Discoidin C2‡
~160 β-sandwich PtdSer (specific) Yes No No 118
ENTH ~150 α-helical solenoid PtdIns(4,5)P2 (some promiscuity)
Yes No No 77
ANTH ~280 α-helical solenoid Phosphoinositides, relatively little specificity
No No Yes 77
BAR ~240 Extended α-helical bundle
Acidic phospholipids (via N-terminal helix)
Yes No Yes 77
F-BAR ~320 Extended α-helical bundle
Acidic phospholipids Unknown No Yes 91, 92
IMD ~250 Extended α-helical bundle
Acidic phospholipids, especially phosphoinositides
Unknown No Yes 97
*It is important to note that functional similarity across a domain class cannot be assumed. For example, up to 80% of pleckstrin homology (PH) domains may not bind phosphoinositides37. Some examples of each domain class may not bind phospholipids at all. ‡Note that the name C2 for these two classes of domain is entirely coincidental. ANTH, AP180 N-terminal homology; BAR, Bin, amphiphysin and Rvs; C1, conserved region-1; C2, conserved region-2; DAG, diacylglycerol; ENTH, epsin N-terminal homology; FYVE, Fab1, YOTB, Vac1, EEA1; Gla, γ-carboxyglutamate-rich; GRAM, glucosyltransferases, Rab-like GTPase activators and myotubularins; IMD, IRSp53/missing-in-metastasis; PH, pleckstrin homology; PKC, protein kinase C; PROPPIN, β-propeller that binds phosphoinositides; PtdIns3P, phosphatidylinositol-3-phosphate; PtdSer, phosphatidylserine; PX, Phox homology.
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Cationic β-groove(second anion-binding site)
Ca2+
PtdSer Membrane
Membrane
PKCα C2 Factor V discoidin C2
Annexin A5 core Prothrombin Gla
Ca2+ Ca2+
Ca2+
groP-Ser Lyso-PtdSer
Zwitterionic phospholipidsA phospholipid with a headgroup that is electrically neutral (no net charge), but that has formal positive and negative charges on different groups. For example, phosphatidylcholine has a positively charged choline headgroup and a negatively charged phosphate. Phosphatidylethanolamine and sphingomyelin are also zwitterionic phospholipids.
short stretches of sequence, often rich in amino acids with basic side chains62. neither specificity nor structural mechanisms of binding are understood. Phosphatidic acid binding promotes localization of some proteins to their sites of action62. by contrast, for some proteins, such as the yeast transcription factor opi1, phosphatidic acid binding seems to sequester the limited quantities of the protein away from its site of action65. opi1 relocates to the nucleus from the endoplasmic reticulum in response to decreased phosphatidic acid levels62,65.
Domains with lower target specificityFor many membrane-binding domains, the physio-logical target is an abundant phospholipid that is ubiquitous in cell membranes (TABLE 2). For example, several phospholipid-binding domains, including C2
domains and annexins, interact with phosphatidylserine. Moreover, many phosphoinositide-binding domains bind similarly to all of the polyphosphoinositides. These facts raise the question as to how specificity — temporal or spatial — can be achieved. Domains with these non-specific characteristics do not simply bind persistently to any membrane that contains phosphatidylserine or polyphosphoinositides. Rather, some of them (such as certain C2 domains and annexins) bind phospholipids only when cytosolic calcium levels are transiently elevated. others, such as bAR and F-bAR domains, seem to interact only with highly curved regions of mem-branes. In contrast to the mechanisms discussed for C1, PH, PX and Fyve domains, these mechanisms allow temporal and spatial specificity in membrane targeting without changing the nature of the headgroup itself.
Ca2+-dependent phosphatidylserine binding: C2 domains. C2 domains, named after the second homology region in PKC, are most well known as Ca2+ sensors and as phosphatidylserine-binding domains66. C2 domains from conventional PKCs are Ca2+-dependent phospho-lipid-binding domains. It is important to stress, however, that not all C2 domains are capable of Ca2+-dependent membrane binding. Indeed, a significant subgroup of C2 domains do not bind Ca2+ at all, and several are now known to bind targets (lipid or protein) other than phosphatidylserine66. C2 domains comprise a character-istic 8-stranded antiparallel β-sandwich of ~130 amino acids, with 3 key inter-strand loops that are responsible for binding both Ca2+ (when relevant) and membranes (FIG. 3). In contrast to PH, PX and Fyve domains, Ca2+-dependent C2 domains lack a basic binding pocket for their negatively charged target lipid. In fact, without bound Ca2+, the canonical C2 domain membrane-binding site is often acidic. bound Ca2+ ions confer positive charge and effectively ‘switch’ the electrostatic characteristics of the binding site so that it can attract negatively charged membranes67. The bound Ca2+ ions also form a ‘bridge’ between the C2 domain and phos-phatidylserine68 (FIG. 3). A combination of these effects explains the Ca2+ dependence of C2-domain binding to phosphatidylserine-containing membranes, although the precise relationship between membrane association and cytosolic Ca2+ levels is not clear for all C2 domains. like other phospholipid-binding domains mentioned above, many (but not all) C2 domains also penetrate the membrane surface66,69.
As mentioned above, lipid selectivity is variable across the large C2 domain family. Ca2+-dependent C2 domains from conventional PKCs specifically recognize phosphati-dylserine with high affinity. Some other Ca2+-dependent C2 domains bind to all anionic phospholipids, and still others prefer zwitterionic phospholipids instead. In addition, some C2 domains reportedly bind selectively to phospho-inositides66, although in a manner that is typically not Ca2+ dependent and that involves a second basic patch that is present on several C2 domains70 (termed the cationic β-groove66) (FIG. 3). Some C2 domains may simultaneously engage multiple membrane components with both their Ca2+-binding loops and cationic β-groove. The diversity in
Figure 3 | Structures of phosphatidylserine-binding domains. The PKCα C2 (Protein Data Bank (PDB) code 1DSY) and annexin A5 core (PDB code 1A8A) domains are intracellular phosphatidylserine (PtdSer)-binding domains. The discoidin C2 and Gla domains are extracellular. The PKCα C2 domain forms a β-sandwich with two Ca2+ ions that are coordinated at one corner, and make bridging interactions with the bound dicaproyl-phosphatidylserine68. Membrane penetration is thought to occur as shown, and the proposed cationic β-groove, at which phosphoinositides are thought to bind C2 domains66, is shown. The annexin core from annexin A5 is shown with bound glycero-phosphorylserine (groP-Ser)72. Ten coordinated Ca2+ ions form bridging interactions between the annexin core and the membrane phospholipids. The discoidin family C2 domain from factor V (PDB code 1CZS) forms a β-sandwich structure, but is not related to the PKC class of C2 domains. Models for membrane binding of the discoidin C2 domain76 involve a basic patch that is formed by several lysine and arginine side chains (shown in stick representation) and a group of aromatic and aliphatic side chains (yellow) that are thought to insert into the membrane. The prothrombin Gla domain (PDB code 1NL2)75 contains seven Ca2+ ions that are coordinated by γ-carboxylglutamates and that are responsible both for stabilizing the Gla-domain structure and for bridging interactions between the Gla domain and the bound lyso-phosphatidylserine (lyso-PtdSer). Significant membrane penetration is also proposed.
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FibrinolysisThe proteolysis of fibrin by plasmin in blood clots.
ProthrombinA pro‑enzyme form of thrombin (also known as factor II), a serine protease that is involved in the blood coagulation cascade by converting fibrinogen into insoluble fibrin.
Factors V, VII, VIII, IX and XCoagulation factors. Factors VII, IX and X are serine protease pro‑enzymes that are involved in the blood coagulation cascade. Once activated, factors V and VIII are cofactors for factor Xa and IXa, respectively.
SynaptotagminAn integral membrane protein with two PKC‑class C2 domains that acts as a Ca2+ sensor in Ca2+‑triggered synaptic vesicle fusion with the plasma membrane.
binding mode, lipid selectivity and Ca2+ dependence of C2 domains raises the intriguing possibility that individual C2 domains are differentially membrane targeted — possibly with defined signalling consequences.
Ca2+-dependent phosphatidylserine binding: annexins. The annexins are also important (and abundant) intracellular Ca2+-dependent phospholipid-binding domains71. They share functional characteristics with PKC C2 domains62, but are structurally unrelated (FIG. 3). The ~310-amino-acid membrane-binding annexin core71 contains 4 annexin repeats, each with 5 α-helices sepa-rated by loops that coordinate Ca2+ ions72. The bound Ca2+ ions form a ‘bridge’ between the membrane surface and the surface of the annexin core domain (FIG. 3) and are coordinated simultaneously by protein and lipid. Additional direct protein–phospholipid interactions (and membrane insertion) are also likely to contribute to membrane binding, but the crucial bridging role of the Ca2+ ions brings membrane association under the tight control of Ca2+ signalling71.
Consistent with this mode of membrane binding, most annexins interact with acidic phospholipids in general, although some bind preferentially to phosphatidyleth-anolamine or phosphatidylcholine instead. Annexin A2 reportedly prefers PtdIns(4,5)P2 (REF. 73). The 13 human annexins have roles in exocytosis, endocytosis, regulation of membrane and/or cytoskeleton interactions and in the control of ion channels71. In addition to their intracellular functions, several annexins have extracellular roles that include stimulating fibrinolysis, inhibiting blood coagu-lation and promoting clearance of apoptotic cells (with extracellularly exposed phosphatidylserine). Intriguingly, annexin A1, A2 and A5 in particular are found both extra- and intracellularly71.
Extracellular phosphatidylserine-binding domains. Two classes of domain interact extracellularly with phosphatidylserine (as the most abundant acidic phos-pholipid) in blood coagulation74. one is the Gla (or γ-carboxyglutamate-rich) domain, which is found in prothrombin as well as in factors VII, IX and X62,74. Another is the discoidin C2 domain, which is found in factors v and vIII and in other proteins such as lactadherin62,74. The discoidin C2 domain is not related to the PKC C2 domain discussed above, despite the unfortunate coinci-dence in nomenclature and some structural resemblance (both are β-sandwiches; FIG. 3).
Gla domains are small (~45 amino acids) and contain 9–12 γ-carboxylated glutamic acid residues that coordi-nate a linear array of Ca2+ ions (FIG. 3) in a largely helical domain75. The bound Ca2+ ions have a crucial ‘bridg-ing’ role in phosphatidylserine binding, as described above for the annexins. Residues with hydrophobic side chains in an adjacent loop also seem to insert into the membrane (FIG. 3).
The ~160-amino-acid discoidin C2 domains do not bind Ca2+, but inter-strand loops in the β-sandwich domain form a positively charged binding site for stereo-specific recognition of phosphatidylserine76. Although the structural architecture of these domains is reminiscent
of their PKC C2 namesakes (FIG. 3), their Ca2+-independent specific phosphatidylserine recognition is more remi-niscent of the way in which PH domains recognize phosphoinositides.
Key roles for membrane topographyA recently identified set of phospholipid-binding domains clearly function beyond simply recruiting their host proteins to the membrane surface. These domains, which include enTH/AnTH, bAR and F-bAR domains77, have little, if any, phospholipid target specificity, and participate in endocytosis, cytokinesis and other processes that involve substantial membrane deformation.
ENTH and ANTH domains. enTH domains were named after a region of epsin n-terminal homology77 that is shared by a family of clathrin adaptor proteins. The AnTH (AP180 n-terminal homology) domain is found in the clathrin adaptor protein AP180 (REFS 78–80). both domains form a superhelical solenoid of α-helices (FIG. 4a), and a similar fold is also found in the membrane-bind-ing n terminus of the AP2 clathrin adaptor α-subunit81. These superhelical folds all bind phosphoinositides, but with relatively little stereospecificity compared with selec-tive PH domains. PtdIns(4,5)P2 is consistently among the preferred ligands78–80 and, as the most abundant phosphoinositide at the plasma membrane (where these proteins function), this is almost certainly the relevant physiological ligand.
AnTH and enTH domains bind differently to PtdIns(4,5)P2. The binding site in the AnTH domain is a surface-lying basic patch that binds PtdIns(4,5)P2 with low affinity79 (FIG. 4a). oligomerization of AP180, conferred by its interaction with clathrin, is thought to allow multiple low-affinity PtdIns(4,5)P2-binding sites to cooperate with one another in driving membrane association of a polymeric complex. by contrast, the PtdIns(4,5)P2-binding site on the enTH domain lies in a well defined pocket78,80. An additional n-terminal amphipathic α-helix (absent from the AnTH domain) becomes ordered upon binding of the enTH domain to PtdIns(4,5)P2 (REF. 78). This helix contacts the lipid head-group — conferring further stereospecificity — but also allows the enTH domain to insert into, and deform, its target membrane82 (FIG. 4a). The membrane deforma-tion that results from enTH- (but not AnTH-) domain binding may help promote membrane invagination at sites of endocytosis77. In principle, any phospholipid-binding domain that penetrates the membrane surface (and there are many) will increase the surface area of the leaflet into which it inserts and thus promote membrane curvature83. The enTH domain represents an extreme example, and one in which membrane recruitment of a protein functions as much to modify the topography of the membrane as to alter localization of the protein. A recent report84 has also suggested that C2 domains in synaptotagmin might promote membrane fusion by inserting into — and bending — membranes in a similar way. This effect is dependent on the presence of multiple C2 domains.
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MembraneMembraneinsertion
P4P4
P5 P5
P1 P1
ENTH
ANTHa
b
~11-nm radius
~14-nm radius
~30-nm radius
~44-nm radius
AmphiphysinN-BAR
Endophilin-A1N-BAR
CIP4F-BAR
IRSp53IMD/I-BAR
Ins(1,4,5)P3
DynaminA large self‑assembling GTPase that plays a crucial role in the scission of endocytic vesicles from the plasma membrane.
Coated pitAn invagination in the plasma membrane, coated with clathrin on its cytoplasmic face, that becomes internalized and forms a clathrin‑coated endocytic vesicle.
BAR and F-BAR domains. Domains in the bAR (‘bin, amphiphysin and Rvs’) family are also thought to promote membrane curvature77. The isolated bAR domain from the endocytic protein amphiphysin binds and tubulates membranes in vitro85 and shows a preference for acidic phospholipids. with its bAR domain promoting the for-mation of membrane tubules, amphiphysin is thought to cooperate with dynamin in defining the necks of coated pits77,85. A bAR domain at the n terminus of endophilin has similar membrane-deforming properties86.
A crystal structure of the bAR domain from D. melanogaster amphiphysin has provided one possible explanation for how it associates with membranes and promotes (or senses) membrane curvature87. The bAR domain adopts an extended coiled-coil structure and forms a long ‘banana shaped’ dimer (FIG. 4b). Peter et al.87 propose that the entire concave face of this dimer (which includes basic patches) abuts the membrane surface. The bAR domain could thus impose membrane curvature, as implied in FIG. 4b, and this could be exacerbated by
the insertion of an important n-terminal amphipathic α-helix into the membrane. even if the proposed elec-trostatic attraction of basic patches on the bAR domain to the negatively charged membrane surface is not suf-ficient to induce membrane curvature, it could allow the bAR domain to function as a curvature ‘sensor’88. If the entire concave surface of the bAR domain must contact the membrane surface for maximum binding affinity (and membrane recruitment), the banana-shaped dimer should bind selectively to (and thus ‘sense’) regions of high membrane curvature87,88. The endophilin bAR domain includes an additional ‘appendage’ that protrudes from the concave face of the banana-shaped dimer (FIG. 4b), and this might alter the preferred curvature and/or penetrate the membrane89,90.
The extended Fer-CIP4 homology (FCH or eFC) domain (now known as F-bAR) is structurally related to bAR domains91,92. F-bAR domains are found in pro-teins from the PCH (Pombe Cdc15 homology) family93, which coordinate membrane–cytoskeleton interactions.
Figure 4 | Structures of phospholipid-binding domains implicated in membrane curvature. a | ENTH and ANTH domains. The epsin ENTH domain (Protein Data Bank (PDB) code 1H0A)78 is shown on the left, and the AP180/CALM ANTH domain is shown on the right (PDB code 1HFA)79 in an equivalent orientation. Both domains consist of an α-helical solenoid, which is C-terminally extended in the longer ANTH domain. Both domains have bound inositol-1,4,5-trisphosphate (Ins(1,4,5)P3). In the ANTH domain, Ins(1,4,5)P3 binds to a surface-lying basic patch that is formed by helices 1 and 2. In the ENTH domain complex, Ins(1,4,5)P3 binds to a different location and makes significant interactions with an N-terminal helix that becomes ordered upon lipid binding (and that is not found in the ANTH domain). This amphipathic helix is thought to penetrate the membrane as shown, with its hydrophobic face (side chains coloured yellow) dissolved in the apolar membrane milieu. b | The BAR domain family. Structures of four different BAR domain (or related) groupings are shown to illustrate how different extended coiled-coil bundles are proposed to ‘sense’ or promote different degrees of membrane curvature77. At the top is the original BAR domain structure from Drosophila melanogaster amphiphysin (PDB code 1URU), the ‘banana-shaped’ nature of which led to the suggestion that these domains sense curvature87. The curvature of the dimer is such that it would follow the surface of a vesicle with a radius of 11 nm. In addition, the N terminus of each molecule is thought to penetrate the membrane, presumably inducing further curvature87. The endophilin-A1 BAR domain (PDB code 1ZWW) is similar, except that it has an appendage in the middle of the concave surface115 that could reduce curvature (or insert into the membrane). The CIP4 (Cdc42-interacting protein-4) F-BAR domain is also shown (PDB code 2EFK)92. This larger domain forms a banana shape with a smaller degree of curvature. Finally, the IMD domain from IRSp53/missing-in-metastasis (PDB code 1Y2O)96 may function as an ‘inverse’ BAR (I-BAR) domain, sensing or stabilizing a curved membrane by lying inside the bend. Side chains that are shown in each structure represent basic side chains that are proposed to interact with the negatively charged membrane surface.
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OligomerizationWeak
Weak
Weak
Weak
Weak
Dimer
Strong
Strong
Strong
Strong
Monomer
Weak
Weak
a
b
c
Two lipids
Lipid in curved membrane
Lipid and protein
ASYNTG
ASYNTG
P P
Protein
Site 2
Site
1Si
te 2
Site
1
Figure 5 | multidomain cooperation. a | Enhancing membrane-binding affinity by domain oligomerization. A phospholipid-binding domain with very low binding affinity will associate with membranes only when it is oligomerized. The two binding sites in the dimer (or oligomer) cooperate with one another to promote a high-avidity interaction. b | Coincidence detection by cooperation of domains in a multidomain protein. Consider a phospholipid-binding domain that specifically recognizes the blue lipid in the figure, but binds too weakly to be able to drive membrane association on its own. If this domain occurs within a protein that also has another domain with similar characteristics (but specificity for the red lipid), then the two domains can cooperate to bind with high avidity to membranes that contain both blue and red lipids (top panel). Similarly, if this domain lies alongside a domain that binds to curved membranes (middle panel), the two domains might cooperate to bind the blue lipid specifically in regions of high curvature. Alternatively, the phospholipid-binding domain might cooperate with a specific (but low affinity) protein-binding domain, such as an SH2 domain, to recruit the multidomain protein specifically to membranes that contain the blue lipid and a tyrosine-phosphorylated protein (bottom panel). c | Coincidence detection by cooperation of binding sites in a single domain. Some domains, such as certain pleckstrin homology (PH) domains, contain binding sites for more than one membrane component. If the two binding sites both bind different lipids, the domain may be selectively recruited to membranes that contain both of these lipids. Alternatively, if the domain binds both a particular type of lipid and a specific membrane-association protein (a small GTPase, for example), then a protein may be recruited selectively to membranes that contain both the lipid and the protein. This mechanism is proposed to explain specific Golgi targeting of certain PH domains103,104.
The ~320-amino-acid F-bAR domain shows some sequence similarity to bAR domains and shares their ability to tubulate membranes and bind acidic phospho-lipids94,95. As shown in FIG. 4b, F-bAR domains form extended coiled-coil dimers that resemble longer and less curved bAR domains, and residues implicated in membrane binding lie on their concave surface. by anal-ogy with the bAR domain hypothesis, F-bAR domains are suggested to sense or induce membrane curvature, but they would favour a smaller degree of curvature than favoured by amphiphysin or endophilin bAR domains (FIG. 4b).
Another bAR-like domain is the IRSp53/missing-in-metastasis (IMD) domain, which structurally resembles a straightened bAR domain96 (FIG. 4b). This domain reportedly binds phosphoinositides and, intriguingly, appears to induce evaginations (rather than invagina-tions) when applied to PtdIns(4,5)P2-containing mem-branes97. This observation is consistent with a role for the IMD domain in filopodia formation and has led to intriguing suggestions that it functions as an ‘inverse bAR domain’97, inducing (or sensing) curvature of the opposite sense (as outlined in FIG. 4b).
Cooperation of multiple interactionsMultivalent multidomain interactions. Many examples of the domains discussed here bind phospholipids too weakly to direct membrane association on their own, but cooperate with other domains in the same protein (or oligomer) to drive multivalent (high avidity) mem-brane binding. one example is the dimeric eeA1 Fyve domain (FIG. 2). Another is the PH domain from the endocytic protein dynamin. The monomeric dynamin PH domain has a millimolar-range Kd for PtdIns(4,5)P2-containing membranes, but simple dimerization brings the apparent Kd into the micromolar range98 (FIG. 5a). As the membrane-binding energies of the two sites in a dimer will be additive, dimerization can in principle reduce the apparent Kd from millimolar to nanomolar values. Such avidity effects might ensure that only dynamin oligomers associate with PtdIns(4,5)P2-containing membranes during endocytic vesicle scis-sion9. Similarly, as mentioned above, vps5 and vps17 (which have low-affinity PX domains) are recruited to PtdIns3P-containing membranes only as part of the multivalent retromer complex57. Such avidity effects can be exploited to control membrane target-ing if oligomerization of a protein with a low-affinity phospholipid-binding domain is tightly regulated.
Multiple domains in the same protein can also cooperate with one another to drive membrane target-ing (FIG. 5b). For example, bAR domains are frequently found alongside other phospholipid-binding domains, such as PH and PX domains87. A PX–bAR protein, for example, might selectively recognize (through multi-domain ‘coincidence detection’99) highly curved regions of PtdIns3P-containing membranes. Indeed, SnX1 reportedly uses this mechanism to target high-curvature membranes emanating from endosomes100. Coincidence detection can also be differentially ‘tuned’, depending on the properties of the cooperating domains. For example,
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FilopodiaThin, transient actin protrusions that extend out from the cell surface and that are formed by the elongation of bundled actin filaments that exist in its core.
SH2 domain(Src homology 2). A small protein domain (110 amino acids) that is found in many signalling proteins and that recognizes phosphorylated tyrosine residues in a particular sequence context. SH2 domains are responsible for recruiting downstream signalling molecules to activated receptor tyrosine kinases at the cell surface.
SH3 domain(Src homology 3). A small protein domain (50–60 amino acids) that recognizes proline‑rich sequences that are important for the assembly of various different signalling complexes.
1. Sheetz, M. P., Sable, J. E. & Dobereiner, H. G. Continuous membrane–cytoskeleton adhesion requires continuous accommodation to lipid and cytoskeleton dynamics. Annu. Rev. Biophys. Biomol. Struct. 35, 417–434 (2006).
2. Di Paolo, G. & De Camilli, P. Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651–657 (2006).
3. Teruel, M. N. & Meyer, T. Translocation and reversible localization of signaling proteins: a dynamic future for signal transduction. Cell 103, 181–184 (2000).
4. Behnia, R. & Munro, S. Organelle identity and the signposts for membrane traffic. Nature 438, 597–604 (2005).
5. McLaughlin, S. & Murray, D. Plasma membrane phosphoinositide organization by protein electrostatics. Nature 438, 605–611 (2005).Provides an important perspective on electrostatic considerations of binding to the membrane surface.
6. McLaughlin, S., Wang, J., Gambhir, A. & Murray, D. PIP2 and proteins: interactions, organization, and information flow. Annu. Rev. Biophys. Biomol. Struct. 31, 151–175 (2002).
7. Stephens, L., McGregor, A. & Hawkins, P. in Biology of Phosphoinositides (ed. Cockcroft, S.) (Oxford Univ. Press, Oxford, UK, 2000).
8. Colon-Gonzalez, F. & Kazanietz, M. G. C1 domains exposed: from diacylglycerol binding to protein–protein interactions. Biochim. Biophys. Acta 1761, 827–837 (2006).This review and references 11, 30, 39, 48, 62, 66 and 77 comprise a valuable set of recent reviews with more detail on each of the phospholipid-binding domains described here.
the RASAl and CAPRI GAPs both contain a PH domain and Ca2+-dependent C2 domains, yet differ dramatically in how their membrane association responds to Ca2+ oscillations101. In CAPRI, PH-domain–membrane inter-actions effectively dampen oscillations in C2-mediated Ca2+-dependent recruitment. In RASAl, oscillatory C2-domain-mediated interactions dominate, which results in a very different time course of membrane recruitment.
The multiple domains that cooperate with one another need not all be phospholipid-binding domains. The domains described here are often found alongside SH2, SH3 and/or other protein-binding domains102. A phospholipid-binding domain and an SH2 domain, for example, might specifically drive a multidomain protein to membranes that contain both the phospholipid target and a specific tyrosine-phosphorylated membrane pro-tein (FIG. 5b). As an illustration, Ras-GAP contains two SH2 domains, a PH domain and a C2 domain — which allows for potentially complex signal integration with input from up to four distinct signals.
Coincidence detection by individual domains. Some of the domains that were described above bind simultane-ously to two targets, and do not require a second domain to function as ‘coincidence detectors’ (FIG. 5c). For exam-ple, the p47phox PX domain and the SoS PH domain both reportedly bind phosphatidic acid in addition to their expected phosphoinositide ligands63,64. Some C2 domains may also bind simultaneously to phosphati-dylserine and other lipids66. Such domains will bind with highest affinity to membranes that contain both of the lipids that they recognize — this is two-lipid coin-cidence detection. There are also examples of phospho-lipid-binding domains with secondary binding sites for specific proteins. The best examples are PH domains from the FAPP1 and oxysterol binding protein (oSbP) family, which specifically recognize Golgi membranes in a way that requires binding to both phosphoinositides and Golgi-localized ARF-family small GTPases103,104. Dual recognition of phospholipids and proteins might be important for specifying the localization of a number of PH and related domains, although structural details remain poorly understood37,38,99.
Conclusions and future directionsThe aim of this review is to provide an overview of phos-pholipid-binding domains and the diverse mechanisms and structures that are used for specifying membrane association. Membrane association of all these domains uses some combination of specific headgroup recogni-tion, delocalized electrostatic attraction to negatively
charged membrane surfaces and penetration of the apolar milieu. Individual domains differ in the extent to which they use these driving forces. both location and timing of membrane association can be tightly control-led. Certain domains — notably some PH, Fyve, PX and C1 domains — associate only with membranes that contain specific lipids. This allows them to be targeted to particular cellular membranes. Moreover, if synthesis of the preferred lipid target is acutely regulated, tempo-ral control of membrane association in cell signalling is also possible — as is the case for PH domains that bind PtdIns(3,4,5)P3. Several other domains are much less specific in the lipids that they recognize, but rely on increases in the level of another second messenger — Ca2+ — to control the timing of their translocation to the membrane.
Although our understanding of the properties of many individual phospholipid-binding domains is now quite sophisticated, our appreciation of how multiple domains in large proteins cooperate with one another remains quite rudimentary. As discussed in the final sections, it is clear that multiple domains (or binding sites) in a protein can cooperate with one another to allow selective binding to phospholipids only in the context of particular lipid mixtures, particular proteins or regions of curvature (FIG. 5b). However, the affinity requirements for bind-ing to the individual targets — and the changes that are required in the levels of those targets — are far from clear. understanding the determinants of these combinatorial ‘codes’ is an important goal for the future.
Another closing thought is underlined by the ques-tion as to whether bAR domains are inducers or sensors of membrane curvature. Similar questions are crucial for all phospholipid-binding domains. we have traditionally focused on the ability of phospholipids to recruit their binding proteins to membranes. However, the converse ability of these domains to recruit phospholipids later-ally within the membrane needs more attention. For example, the polybasic myristoylated alanine-rich PKC substrate (MARCKS) peptide clusters PtdIns(4,5)P2 once it has associated with negatively charged membranes5,6. In the context of multidomain proteins, it seems likely that several of the domains discussed in this review might be as important for modulating lipid distribution in membranes to which they are apposed as they are for altering the subcellular localization of the proteins in which they are found. Transient membrane target-ing that allows dynamic redistribution of lipids at key locations by phospholipid-binding domains could be important in processes such as membrane fission and vesicle scission, for example.
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9. Lemmon, M. A. & Ferguson, K. M. Signal-dependent membrane targeting by pleckstrin homology (PH) domains. Biochem. J. 350, 1–18 (2000).
10. Oancea, E., Teruel, M. N., Quest, A. F. & Meyer, T. Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells. J. Cell Biol. 140, 485–498 (1998).
11. Várnai, P. & Balla, T. Live cell imaging of phosphoinositide dynamics with fluorescent protein domains. Biochim. Biophys. Acta 1761, 957–967 (2006).
12. Zhang, G., Kazanietz, M. G., Blumberg, P. M. & Hurley, J. H. Crystal structure of the Cys2 activator-binding domain of protein kinase Cδ in complex with phorbol ester. Cell 81, 917–924 (1995).One of the earliest structural views of how a specific membrane-targeting domain functions.
13. Kazanietz, M. G., Barchi, J. J., Omichinski, J. G. & Blumberg, P. M. Low affinity binding of phorbol esters to protein kinase C and its recombinant cysteine-rich region in the absence of phospholipids J. Biol. Chem. 270, 14679–14684 (1995).
14. Harlan, J. E., Hajduk, P. J., Yoon, H. S. & Fesik, S. W. Pleckstrin homology domains bind to phosphatidylinositol-4,5-bisphosphate. Nature 371, 168–170 (1994).First report that some PH domains can bind phosphoinositides.
15. Lemmon, M. A., Ferguson, K. M., O’Brien, R., Sigler, P. B. & Schlessinger, J. Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain. Proc. Natl Acad. Sci. USA 92, 10472–10476 (1995).
16. Garcia, P. et al. The pleckstrin homology domain of phospholipase C-δ1 binds with high affinity to phosphatidylinositol 4,5-bisphosphate in bilayer membranes. Biochemistry 34, 16228–16234 (1995).
17. Hirose, K., Kadowaki, S., Tanabe, M., Takeshima, H. & Iino, M. Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns. Science 284, 1527–1530 (1999).
18. Kavran, J. M. et al. Specificity and promiscuity in phosphoinositide binding by pleckstrin homology domains. J. Biol. Chem. 273, 30497–30508 (1998).
19. Ferguson, K. M., Lemmon, M. A., Schlessinger, J. & Sigler, P. B. Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain. Cell 83, 1037–1046 (1995).First structural view of specific phosphoinositide binding by a protein domain.
20. Flesch, F. M., Yu, J. W., Lemmon, M. A. & Burger, K. N. C. Membrane activity of the phospholipase C-δ1 pleckstrin homology (PH) domain. Biochem. J. 389, 435–441 (2005).
21. Cantley, L. C. The phosphoinositide 3-kinase pathway. Science 296, 1655–1657 (2002).
22. Fukuda, M., Kojima, T., Kabayama, H. & Mikoshiba, K. Mutation of the pleckstrin homology domain of Bruton’s tyrosine kinase in immunodeficiency impaired inositol 1,3,4,5-tetrakisphosphate binding capacity. J. Biol. Chem. 271, 30303–30306 (1996).
23. Salim, K. et al. Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton’s tyrosine kinase. EMBO J. 15, 6241–6250 (1996).
24. Klarlund, J. K. et al. Signaling by phosphoinositide-3,4,5-trisphosphate through proteins containing pleckstrin and Sec7 homology domains. Science 275, 1927–1930 (1997).
25. Franke, T. F., Kaplan, D. R., Cantley, L. C. & Toker, A. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 275, 665–668 (1997).References 24 and 25 were instrumental in establishing PH domains as effector domains of PI3K signalling.
26. Venkateswarlu, K., Oatey, P. B., Tavare, J. M. & Cullen, P. J. Insulin-dependent translocation of ARNO to the plasma membrane of adipocytes requires phosphatidylinositol 3-kinase. Curr. Biol. 8, 463–466 (1998).
27. Lindvall, J. M. et al. Bruton’s tyrosine kinase: cell biology, sequence conservation, mutation spectrum, siRNA modifications, and expression profiling. Immunol. Rev. 203, 200–215 (2005).
28. Carpten, J. D. et al. A transforming mutation in the pleckstrin homology domain of AKT1 in cancer. Nature 448, 439–444 (2007).Describes important pathological consequences of the promotion of aberrant membrane association by a PH domain.
29. Huang, Y. H. et al. Positive regulation of Itk PH domain function by soluble IP4. Science 316, 886–889 (2007).
30. DiNitto, J. P. & Lambright, D. G. Membrane and juxtamembrane targeting by PH and PTB domains. Biochim. Biophys. Acta 1761, 850–867 (2006).
31. Isakoff, S. J. et al. Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast. EMBO J. 17, 5374–5387 (1998).
32. Ferguson, K. M. et al. Structural basis for discrimination of 3-phosphoinositides by pleckstrin homology domains. Mol. Cell 6, 373–384 (2000).
33. Ceccarelli, D. F. et al. Non-canonical interaction of phosphoinositides with pleckstrin homology domains of Tiam1 and ArhGAP9. J. Biol. Chem. 282, 13864–13874 (2007).
34. Hyvönen, M. et al. Structure of the binding site for inositol phosphates in a PH domain. EMBO J. 14, 4676–4685 (1995).
35. Slagsvold, T. et al. Eap45 in mammalian ESCRT-II binds ubiquitin via a phosphoinositide-interacting GLUE domain. J. Biol. Chem. 280, 19600–19606 (2005).
36. Teo, H. et al. ESCRT-I core and ESCRT-II GLUE domain structures reveal role for GLUE in linking to ESCRT-I and membranes. Cell 125, 99–111 (2006).Structurally illustrates an unexpected occurrence of a split PH domain that also binds phosphoinositides.
37. Yu, J. W. et al. Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains. Mol. Cell 13, 677–688 (2004).Provides a valuable perspective on the likely diversity of PH domain function.
38. Lemmon, M. A. Pleckstrin homology domains: not just for phosphoinositides. Biochem. Soc. Trans. 32, 707–711 (2004).
39. Kutateladze, T. G. Phosphatidylinositol 3-phosphate recognition and membrane docking by the FYVE domain. Biochim. Biophys. Acta 1761, 868–877 (2006).
40. Misra, S. & Hurley, J. H. Crystal structure of a phosphatidylinositol 3-phosphate-specific membrane-targeting motif, the FYVE domain of Vps27p. Cell 97, 657–666 (1999).
41. Dumas, J. J. et al. Multivalent endosome targeting by homodimeric EEA1. Mol. Cell 8, 947–958 (2001).
42. Kutateladze, T. & Overduin, M. Structural mechanism of endosome docking by the FYVE domain. Science 291, 1793–1796 (2001).
43. Stahelin, R. V. et al. Phosphatidylinositol 3-phosphate induces the membrane penetration of the FYVE domains of Vps27p and Hrs. J. Biol. Chem. 277, 26379–26388 (2002).
44. Gillooly, D. J. et al. Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J. 19, 4577–4588 (2000).
45. Hayakawa, A. et al. Structural basis for endosomal targeting by FYVE domains. J. Biol. Chem. 279, 5958–5966 (2004).
46. Ponting, C. P. Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3-kinases: binding partners of SH3 domains? Plant Prot. Sci. 5, 2353–2357 (1996).
47. Ellson, C. D., Andrews, S., Stephens, L. R. & Hawkins, P. T. The PX domain: a new phosphoinositide-binding module. J. Cell Sci. 115, 1099–1105 (2002).
48. Seet, L. F. & Hong, W. The Phox (PX) domain proteins and membrane traffic. Biochim. Biophys. Acta 1761, 878–896 (2006).
49. Yu, J. W. & Lemmon, M. A. All Phox homology (PX) domains from Saccharomyces cerevisiae specifically recognize phosphatidylinositol 3-phosphate. J. Biol. Chem. 276, 44179–44184 (2001).
50. Kanai, F. et al. The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nature Cell Biol. 3, 675–678 (2001).
51. Song, X. et al. Phox homology domains specifically bind phosphatidylinositol phosphates. Biochemistry 40, 8940–8944 (2001).
52. Bravo, J. et al. The crystal structure of the PX domain from p40phox bound to phosphatidylinositol 3-phosphate. Mol. Cell 8, 829–839 (2001).
53. Ellson, C. D. et al. PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40phox. Nature Cell Biol. 3, 679–682 (2001).
54. Xu, Y., Hortsman, H., Seet, L., Wong, S. H. & Hong, W. SNX3 regulates endosomal function through its PX-domain-mediated interaction with PtdIns(3)P. Nature Cell Biol. 3, 658–666 (2001).
55. Zhong, Q. et al. Determinants of the endosomal localization of sorting nexin 1. Mol. Biol. Cell 16, 2049–2057 (2005).
56. Seaman, M. N., McCaffery, J. M. & Emr, S. D. A membrane coat complex essential for endosome- to-Golgi retrograde transport in yeast. J. Cell Biol. 142, 665–681 (1998).
57. Seaman, M. N. & Williams, H. P. Identification of the functional domains of yeast sorting nexins Vps5p and Vps17p. Mol. Biol. Cell 13, 2826–2840 (2002).
58. Michell, R. H., Heath, V. L., Lemmon, M. A. & Dove, S. K. Phosphatidylinositol 3,5-bisphosphate: metabolism and cellular functions. Trends Biochem. Sci. 31, 52–63 (2006).
59. Dove, S. K. et al. Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors. EMBO J. 23, 1922–1933 (2004).
60. Jeffries, T. R., Dove, S. K., Michell, R. H. & Parker, P. J. PtdIns-specific MPR pathway association of a novel WD40 repeat protein, WIPI49. Mol. Biol. Cell 15, 2652–2663 (2004).
61. Stromhaug, P. E., Reggiori, F., Guan, J., Wang, C. W. & Klionsky, D. J. Atg21 is a phosphoinositide binding protein required for efficient lipidation and localization of Atg8 during uptake of aminopeptidase I by selective autophagy. Mol. Biol. Cell 15, 3553–3566 (2004).
62. Stace, C. L. & Ktistakis, N. T. Phosphatidic acid- and phosphatidylserine-binding proteins. Biochim. Biophys. Acta 1761, 913–926 (2006).Provides a valuable review of the characteristics of phosphatidic acid-binding sequences.
63. Zhao, C., Du, G., Skowronek, K., Frohman, M. A. & Bar-Sagi, D. Phospholipase D2-generated phosphatidic acid couples EGFR stimulation to Ras activation by Sos. Nature Cell Biol. 9, 706–712 (2007).
64. Karathanassis, D. et al. Binding of the PX domain of p47phox to phosphatidylinositol 3,4-bisphosphate and phosphatidic acid is masked by an intramolecular interaction. EMBO J. 21, 5057–5068 (2002).
65. Loewen, C. J. et al. Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid. Science 304, 1644–1647 (2004).Describes an intriguing mechanism of transcription-factor inhibition by sequestration at specific extranuclear membranes.
66. Cho, W. & Stahelin, R. V. Membrane binding and subcellular targeting of C2 domains. Biochim. Biophys. Acta 1761, 838–849 (2006).
67. Murray, D. & Honig, B. Electrostatic control of the membrane targeting of C2 domains. Mol. Cell 9, 145–154 (2002).
68. Verdaguer, N., Corbalan-Garcia, S., Ochoa, W. F., Fita, I. & Gomez-Fernandez, J. C. Ca2+ bridges the C2 membrane-binding domain of protein kinase Cα directly to phosphatidylserine. EMBO J. 18, 6329–6338 (1999).
69. Davletov, B., Perisic, O. & Williams, R. L. Calcium-dependent membrane penetration is a hallmark of the C2 domain of cytosolic phospholipase A2 whereas the C2A domain of synaptotagmin binds membranes electrostatically. J. Biol. Chem. 273, 19093–19096 (1998).
70. Sánchez-Bautista, S., Marin-Vicente, C., Gómez-Fernandez, J. C. & Corbalán-Garcia, S. The C2 domain of PKCα is a Ca2+-dependent PtdIns(4,5)P2 sensing domain: a new insight into an old pathway. J. Mol. Biol. 362, 901–914 (2006).
71. Gerke, V., Creutz, C. E. & Moss, S. E. Annexins: linking Ca2+ signalling to membrane dynamics. Nature Rev. Mol. Cell Biol. 6, 449–461 (2005).
72. Swairjo, M. A., Concha, N. O., Kaetzel, M. A., Dedman, J. R. & Seaton, B. A. Ca2+-bridging mechanism and phospholipid head group recognition in the membrane-binding protein annexin V. Nature Struct. Biol. 2, 968–974 (1995).
73. Rescher, U., Ruhe, D., Ludwig, C., Zobiack, N. & Gerke, V. Annexin 2 is a phosphatidylinositol (4,5)-bisphosphate binding protein recruited to actin assembly sites at cellular membranes. J. Cell Sci. 117, 3473–3480 (2004).
74. Zwaal, R. F., Comfurius, P. & Bevers, E. M. Lipid–protein interactions in blood coagulation. Biochim. Biophys. Acta 1376, 433–453 (1998).
75. Huang, M. et al. Structural basis of membrane binding by Gla domains of vitamin K-dependent proteins. Nature Struct. Biol. 10, 751–756 (2003).
R E V i E W s
110 | FebRuARy 2008 | voluMe 9 www.nature.com/reviews/molcellbio
R E V i E W s
© 2008 Nature Publishing Group
76. Macedo-Ribeiro, S. et al. Crystal structures of the membrane-binding C2 domain of human coagulation factor V. Nature 402, 434–439 (1999).
77. Itoh, T. & De Camilli, P. BAR, F-BAR (EFC) and ENTH/ANTH domains in the regulation of membrane-cytosol interfaces and membrane curvature. Biochim. Biophys. Acta 1761, 897–912 (2006).
78. Ford, M. G. et al. Curvature of clathrin-coated pits driven by epsin. Nature 419, 361–366 (2002).One of the first studies to link phosphoinositide-binding proteins to the possible induction of membrane curvature.
79. Ford, M. G. et al. Simultaneous binding ofSimultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science 291, 1051–1055 (2001).
80. Itoh, T. et al. Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis. Science 291, 1047–1051 (2001).
81. Collins, B. M., McCoy, A. J., Kent, H. M., Evans, P. R. & Owen, D. J. Molecular architecture and functional model of the endocytic AP2 complex. Cell 109, 523–535 (2002).
82. Stahelin, R. V. et al. Contrasting membrane interaction mechanisms of AP180 N-terminal homology (ANTH) and epsin N-terminal homology (ENTH) domains. J. Biol. Chem. 278, 28993–28999 (2003).
83. Zimmerberg, J. & Kozlov, M. M. How proteins produce cellular membrane curvature. Nature Rev. Mol. Cell Biol. 7, 9–19 (2006).Valuable perspectives on physical processes that are involved in inducing membrane curvature.
84. Martens, S., Kozlov, M. M. & McMahon, H. T. How synaptotagmin promotes membrane fusion. Science 316, 1205–1208 (2007).
85. Takei, K., Slepnev, V. I., Haucke, V. & De Camilli, P. Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nature Cell Biol. 1, 33–39 (1999).
86. Farsad, K. et al. Generation of high curvature membranes mediated by direct endophilin bilayer interactions. J. Cell Biol. 155, 193–200 (2001).
87. Peter, B. J. et al. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495–499 (2004).First structure of a BAR domain, which led to the current notion that these are sensors (or inducers) of membrane curvature.
88. Zimmerberg, J. & McLaughlin, S. Membrane curvature: how BAR domains bend bilayers. Curr. Biol. 14, R250–R252 (2004).
89. Masuda, M. et al. Endophilin BAR domain drives membrane curvature by two newly identified structure-based mechanisms. EMBO J. 25, 2889–2897 (2006).
90. Gallop, J. L. et al. Mechanism of endophilin N-BAR domain-mediated membrane curvature. EMBO J. 25, 2898–2910 (2006).
91. Henne, W. M. et al. Structure and analysis of FCHo2 F-BAR domain: a dimerizing and membrane recruitment module that effects membrane curvature. Structure 15, 839–852 (2007).
92. Shimada, A. et al. Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis. Cell 129, 761–772 (2007).
93. Chitu, V. & Stanley, E. R. Pombe Cdc15 homology (PCH) proteins: coordinators of membrane–cytoskeletal interactions. Trends Cell Biol. 17, 145–156 (2007).
94. Itoh, T. et al. Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev. Cell 9, 791–804 (2005).
95. Tsujita, K. et al. Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis. J. Cell Biol. 172, 269–279 (2006).
96. Millard, T. H. et al. Structural basis of filopodia formation induced by the IRSp53/MIM homology domain of human IRSp53. EMBO J. 24, 240–250 (2005).
97. Mattila, P. K. et al. Missing-in-metastasis and IRSp53 deform PI(4,5)P2-rich membranes by an inverse BAR domain-like mechanism. J. Cell Biol. 176, 953–964 (2007).
98. Klein, D. E., Lee, A., Frank, D. W., Marks, M. S. & Lemmon, M. A. The pleckstrin homology domains of dynamin isoforms require oligomerization for high affinity phosphoinositide binding. J. Biol. Chem. 273, 27725–27733 (1998).
99. Carlton, J. G. & Cullen, P. J. Coincidence detection in phosphoinositide signaling. Trends Cell Biol. 15, 540–547 (2005).Informative survey of mechanisms for coincidence detection in membrane binding.
100. Carlton, J. et al. Sorting nexin-1 mediates tubular endosome-to-TGN transport through coincidence sensing of high-curvature membranes and 3-phosphoinositides. Curr. Biol. 14, 1791–1800 (2004).
101. Liu, Q. et al. CAPRI and RASAL impose different modes of information processing on Ras due to contrasting temporal filtering of Ca2+. J. Cell Biol. 170, 183–190 (2005).
102. Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 103, 211–225 (2000).
103. Godi, A. et al. FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P. Nature Cell Biol. 6, 393–404 (2004).
104. Levine, T. P. & Munro, S. Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components. Curr. Biol. 12, 695–704 (2002).One of the first papers to suggest that certain PH domains may bind simultaneously to phospholipid and protein targets.
105. Tanford, C. The Hydrophobic Effect (Wiley, New York, 1980).
106. Stephens, L. R., Jackson, T. R. & Hawkins, P. T. Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate: a new intracellular signalling system? Biochim. Biophys. Acta 1179, 27–75 (1993).
107. Morris, J. B., Hinchliffe, K. A., Ciruela, A., Letcher, A. J. & Irvine, R. F. Thrombin stimulation of platelets causes an increase in phosphatidylinositol 5-phosphate revealed by mass assay. FEBS Lett. 475, 57–60 (2000).
108. Pendaries, C. et al. Emerging roles of phosphatidylinositol monophosphates in cellular signaling and trafficking. Adv. Enzyme Regul. 45, 201–214 (2005).
109. Mitra, P. et al. A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast. J. Cell Biol. 166, 205–211 (2004).
110. Maffucci, T., Brancaccio, A., Piccolo, E., Stein, R. C. & Falasca, M. Insulin induces phosphatidylinositol-3- phosphate formation through TC10 activation. EMBO J. 22, 4178–4189 (2003).
111. Pettitt, T. R., Dove, S. K., Lubben, A., Calaminus, S. D. & Wakelam, M. J. Analysis of intact phosphoinositides in biological samples. J. Lipid Res. 47, 1588–1596 (2006).
112. Narayan, K. & Lemmon, M. A. Determining selectivity of phosphoinositide-binding domains. Methods 39, 122–133 (2006).
113. Dowler, S. et al. Identification of pleckstrin-homology- domain-containing proteins with novel phosphoinositide-binding specificities. Biochem. J. 351, 19–31 (2000).
114. Gozani, O. et al. The PHD finger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor. Cell 114, 99–111 (2003).
115. Weissenhorn, W. Crystal structure of the endophilin-A1 BAR domain. J. Mol. Biol. 351, 653–661 (2005).
116. Letunic, I. et al. SMART 5: domains in the context of genomes and networks. Nucleic Acids Res. 34, D257–D260 (2006).
117. Bhattacharyya, R. P., Remenyi, A., Yeh, B. J. & Lim, W. A. Domains, motifs, and scaffolds: the role of modular interactions in the evolution and wiring of cell signaling circuits. Annu. Rev. Biochem. 75, 655–680 (2006).
118. Fuentes-Prior, P., Fujikawa, K. & Pratt, K. P. New insights into binding interfaces of coagulation factors V and VIII and their homologues — lessons from high resolution crystal structures. Curr. Protein Pept. Sci. 3, 313–339 (2002).
AcknowledgementsI thank K. Ferguson and members of the Lemmon laboratory for comments on this review. Work in this area in my labora-tory is funded by the National Institute of General Medical Sciences (NIGMS).
DATABASESRcsB protein data Bank: http://www.rcsb.org/pdb/home/home.do 1A8A | 1CZS | 1DSY | 1H6H | 1H0A | 1HFA | 1JOC | 1MAI | 1NL2 | 1O7K | 1PTR | 1URU | 1Y2O | 1ZWW | 2EFK | 2P0DuniprotKB: http://ca.expasy.org/sprot AP180 | ARHGAP9 | TIAM1 | VPS36
FURTHER INFORMATIONMark A. lemmon’s homepage: http://www.med.upenn.edu/camb/faculty/cbp/lemmon.htmlsMART (simple modular architecture research tool):http://smart.embl-heidelberg.deMembrane targeting domains resource (university of illinois at chicago):http://proteomics.bioengr.uic.edu/metador/MeTaDoR.htmlstructure gallery, Roger Williams laboratory web site:http://www.mrc-lmb.cam.ac.uk/rlw/text/structuregallery.htmlHarvey McMahon’s laboratory web site:http://www.endocytosis.orgJim Hurley’s laboratory web site:http://www-mslmb.niddk.nih.gov/hurleygroup.html
all linkS are acTive in THe online PDF
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nATuRe RevIewS | molecular cell biology voluMe 9 | FebRuARy 2008 | 111
f o c u s o n l i p i d s
© 2008 Nature Publishing Group
