Immunogenetics (2003) 54:817–829DOI 10.1007/s00251-002-0534-9

R E V I E W

Esther van de Vosse · Elgin G. R. Lichtenauer-Kaligis ·Jaap T. van Dissel · Tom H. M. Ottenhoff

Genetic variations in the interleukin-12/interleukin-23receptor (b1) chain, and implications for IL-12and IL-23 receptor structure and functionReceived: 9 December 2002 / Accepted: 9 December 2002 / Published online: 21 February 2003� Springer-Verlag 2003

Abstract Cell-mediated immunity (CMI) plays an essen-tial role in human host defense against intracellularbacteria. Type-1 cytokines, particularly gamma interferon(IFN-g), interleukin-12 (IL-12), and IL-23, the majorcytokines that regulate IFN-g production, are essential inCMI. This is illustrated by patients with unusual severeinfections caused by poorly pathogenic mycobacteria andSalmonella species, in whom genetic deficiencies havebeen identified in several key genes in the type-1 cytokinepathway, including IL12RB1, the gene encoding the b1chain of the IL-12 and IL-23 receptors. Several mutationsin IL12RB1 with deleterious effects on human IL-12Rfunction have been identified, including nonsense andmissense mutations. In addition, a number of codingIL12RB1 polymorphisms have been reported. In order togain more insight into the effect that IL12RB1 mutationsand genetic variations can have on IL-12Rb1 function,three approaches have been followed. First, we deter-mined the degree of conservation at the variant aminoacid positions in IL-12Rb1 between different species,using known deleterious mutations, known variations inIL-12Rb1, as well as novel coding variations that we haveidentified at position S74R and R156H. Second, weanalyzed the potential impact of these amino acidvariations on the three-dimensional structure of the IL-12Rb1 protein. Third, we analyzed the putative functionsof different IL-12Rb1 domains, partly based on theirhomology with gp130, and analyzed the possible effectsof the above amino acid variations on the function ofthese domains. Based on these analyses, we propose an

integrated model of IL-12Rb1 structure and function.This significantly enhances our molecular understandingof the human IL-12 and IL-23 systems.

Keywords Interleukin-12 · Interleukin-23 ·Interleukin-12Rb1 · Gamma interferon · Human

Introduction

Effective cell-mediated immunity plays an essential rolein human host defense against intracellular bacteria,including mycobacteria and salmonellae, and is criticallydependent on type-1 cytokines. Upon infection withintracellular bacteria, antigen presenting cells (APCs)such as macrophages and dendritic cells produce inter-leukin-12 (IL-12) (D’Andrea et al. 1992; Taoufik et al.2001) (Fig. 1) as well as three other cytokines withactivities similar to IL-12, particularly IL-18 (Nakanishiet al. 2001), IL-23 (Oppmann et al. 2000) and IL-27(Pflanz et al. 2002). Bioactive IL-12 (IL-12p70) is aheterodimer consisting of two disulfide-bonded glycopro-tein subunits, a p40 and a p35 chain. IL-12 binds to IL-12receptor complexes (IL-12R) on the surface of T helper-1(Th-1) and natural killer (NK) cells and thus stimulatescellular proliferation and production of gamma interferon(IFN-g), a key effector molecule in cell-mediated immu-nity (Chan et al. 1991; Gately et al. 1994; Kobayashi et al.1989; Murphy et al. 1994; Wolf et al. 1991). IFN-g in turnbinds to the IFN-g receptor (IFN-gR) on macrophages anddendritic cells and activates transcription of genes thatcontribute to the killing of the intracellular bacteria (Bachet al. 1997). The role of IL-12 has been extensivelyreviewed by Trinchieri and Scott (1999).

The IL-12-like cytokine IL-23 consists of an IL-12p40subunit, disulfide-linked to a unique p19 subunit. Expres-sion of IL-23 is high in Th-1 cells, activated macrophagesand dendritic cells (Oppmann et al. 2000). The IL-23p19subunit is most closely related to the p35 subunit of IL-12and, accordingly, IL-23 appears to have a very similarfunction to IL-12.

E. van de Vosse · E. G. R. Lichtenauer-Kaligis · J. T. van DisselDepartment of Infectious Diseases,Leiden University Medical Center (LUMC),Leiden, The Netherlands

T. H. M. Ottenhoff ())Department of Immunohematology and Blood Transfusion,Bldg 1, L3–33, Leiden University Medical Center (LUMC),Albinusdreef 2, PO Box 9600, 2300 RC Leiden, The Netherlandse-mail: t.h.m.ottenhoff@lumc.nlTel.: +31-71-5265128Fax: +31-71-5216751

In patients with unusual, severe infections caused byotherwise poorly pathogenic mycobacteria and Salmonel-la species, genetic deficiencies have been identified inseveral key genes in the type-1 cytokine pathway. Thesedefects lead to a strongly impaired capacity to eitherproduce or respond to IFN-g. Many of these patientspresented at infancy with disseminated Mycobacteriumbovis BCG infection following vaccination with bacilleCalmette-Gu�rin (BCG). A first major group of thesepatients with type-1 cytokine pathway defects haddeleterious mutations in the gene encoding the IFN-gR1chain of the IFN-g receptor (reviewed by Ottenhoff et al.2002). A second major group of patients with similarsyndromes had genetic defects in the IL-12Rb1 chain ofthe IL-12 receptor (Aksu et al. 2001; Altare et al. 1998a,2001; de Jong et al. 1998; Elloumi-Zghal et al. 2002;Lichtenauer-Kaligis et al. 2003; Sakai et al. 2001;Verhagen et al. 2000). A third group was identified withdefects in IL-12p40 (Altare et al. 1998b; Elloumi-Zghal etal. 2002; Picard et al. 2002). Finally, a minority ofpatients had defects in the IFN-gR2 chain (Dorman andHolland 1998; D�ffinger et al. 2000; Holland 2001) or inthe IFN-gR associated signal transducer and activator oftranscription (STAT1) (Dupuis et al. 2001). The clinicaloutcome of this range of genetic deficiencies has been

reviewed extensively by Casanova and Abel (2002) andOttenhoff and co-workers (1998, 2002).

The IL-12Rb1 subunit in the IL-12Rand the IL-23R complex

The majority of deleterious mutations in IL12RB1 iden-tified in patients with genetic IL-12Rb1 deficiency lead topremature truncation of the protein, either directly byencoding a premature stop codon prior to the transmem-brane region, or indirectly as a result of frameshiftmutations. All these mechanisms account for the absenceof IL-12Rb1 expression and function in these patients. Ina minority of patients, complete IL-12Rb1 deficiencyresulted from missense mutations in IL-12Rb1, andrecently a first case with partial IL-12Rb1 deficiencyresulting from a missense mutation (C198R) was de-scribed (Lichtenauer-Kaligis et al. 2003). All thesemissense mutations in IL12RB1 precluded detectableexpression of cell surface IL-12Rb1 protein, althoughnone affected the transmembrane domain or signalpeptide (Aksu et al. 2001; Altare et al. 2001; Lichte-nauer-Kaligis et al. 2003; Sakai et al. 2001). Moreover,several coding IL-12Rb1 polymorphisms have been

Fig. 1 The type-1 cytokine pathway. IFN-g binds to IFN-gR1/R2-receptor complexes on mononuclear phagocytes. IFN-g, togetherwith TNF-a, activates microbicidal mechanisms in macrophages.Production of IFN-g is regulated by IL-12p70, which binds to IL-12Rb1/b2-complexes on Th1- and NK-cells. IL-12R signalsthrough IL-12Rb2-associated Stat4, involving also Jak2, Tyk2,

Stat1, Stat3 and Stat5. In addition to IL-12, also IL-23, IL-18, IL-27, and possibly other molecules like TCCR-ligand, interferon-aand chemokines, are involved in inducing IFN-g production.Mutations that lead to enhanced susceptibility to intracellularinfection have been identified in IFN-gR1, IFN-gR2, IL-12Rb1, IL-12p40, and Stat1

818

described that do not result in loss-of-function phenotypes[(Elloumi-Zghal et al. 2002; Sakai et al. 2001) and ourdata described here].

Since IL-12Rb1 is an essential component of IL-12Ras well as IL-23R (Ottenhoff et al. 2002), IL-12Rb1deficiencies will affect both IL-12R and IL-23R depen-dent, innate and adaptive host immunity. IL-12 and IL-23have similar but not identical effects. They both activatetranscription of IFN-g through activation of STATs(Belladonna et al. 2002; Oppmann et al. 2000) and bothenhance production of IL-12p70, the combination of thetwo resulting in an additive effect (Belladonna et al.2002). However, IL-23 induces proliferation of memory Tcells and has little or no effect on naive T cells, whereasIL-12 has an effect on both cell types (Oppmann et al.2000). In order to better understand the biology of the IL-12 and IL-23 systems, it is important to analyze thestructure-function relationship of IL-12Rb1. In this studywe summarize and discuss recent insights into thestructure and function of the domains that are present inIL-12Rb1, and carefully examine the effects that varia-tions in the amino acid sequence may have on thestructure of the IL-12Rb1 protein and its function in IL-12/IL-12R and IL-23/IL-23R signaling. We also reporttwo novel genetic variations in the IL-12Rb1 protein.

The IL12RB1 gene was cloned based on its affinity forIL-12 (Chua et al. 1994) and appeared to be highly relatedto a subgroup of the hemopoietin receptor superfamilythat included gp130, G-CSFR and LIF-R, with a highestsimilarity to gp130, which is a signal-transducing receptorfor a family of cytokines. It was shown that IL-12Rb1 byitself had a low affinity for IL-12 (Chua et al. 1994) andan additional receptor subunit, IL-12Rb2, was subse-quently identified (Presky et al. 1996; van Rietschoten etal. 2000). Human IL-12Rb1 and IL-12Rb2 separatelybind IL-12 with low affinity, but together bind IL-12 withhigh affinity.

IL-23 binds to IL-12Rb1, but not IL-12Rb2 (Oppmannet al. 2000), suggesting that an additional IL-23 receptorchain is required. Recently, this chain was identified byselecting for IL-23 binding in co-expression experimentswith IL-12Rb1. We will refer to this IL-23R receptorchain as IL-23Rb3 and to the receptor complex as IL-23R.

As indicated in detail in Fig. 2, the IL-12Rb1 proteinconsists of 662 amino acids (aa), with a 24-aa N-terminalsignal peptide, giving a mature protein of 638 aa. IL-12Rb1 does not contain the N-terminal Ig-like activationdomain that is present in gp130, nor does it contain thecytoplasmic tyrosine residues that are required fordocking and activating STAT4. It therefore lacks asignal-transducing domain. In comparison with IL-12Rb1, IL-12Rb2 has a higher homology to gp130 andcontains both the N-terminal Ig-like activation domainand the cytoplasmic tyrosine residues (Presky et al. 1996),indicating that IL-12Rb2 does contain a signal-transduc-ing domain. The IL-23Rb3 protein is very similar to IL-12Rb2, although it does not contain the three fibronectintype III domains adjacent to the transmembrane domain

that are present in both IL-12Rb2 and IL-12Rb1. Like IL-12Rb2, IL-23Rb3 contains a signal-transducing domain.

Interaction of IL-12 with IL-12R

IL-12 appears to interact with IL-12Rb1 primarily viadomains on the IL-12p40 subunit and with IL-12Rb2 viaa p40/p35 heterodimeric specific region of IL-12 (Preskyet al. 1998). Receptor heterodimers often have a modulararchitecture in which one subunit, the ‘driver’, defines theexact signaling, whereas the other subunit is a relativelynon-specific ‘trigger’ element in the initiation of signaling(Lai et al. 1996). In such a model the IL-12Rb2 subunitwould be the ‘driver’ and the IL-12Rb1 subunit would bethe ‘trigger’.

Signaling through IL-12R is mediated by cytoplasmicTYK2 and JAK2, members of the JAK family of proteintyrosine kinases that are tyrosine phosphorylated whenIL-12 binds to the IL-12R complex. TYK2 directlyinteracts with IL-12Rb1, whereas JAK2 can be phos-phorylated by both IL-12R subunits, although directinteraction has only been found with IL-12Rb2 (Zou et al.1997). The JAKs in turn are necessary for the tyrosinephosphorylation and activation of STAT4 that is bound toIL-12Rb2 (Naeger et al. 1999; Yao et al. 1999), afterwhich STAT4 homodimerizes, translocates to the nucleusand binds to its target DNA to activate transcription ofIFN-g and other target genes (Bacon et al. 1995). IL-12stimulation also activates STAT1, STAT3 and STAT5(Gollob et al. 1998; Jacobson et al. 1995), although directinteraction between STAT1, STAT3 or STAT5 and eitherof the IL-12R chains has not yet been demonstrated.

Deleterious mutations in IL-12Rb1

To date, 21 patients in 17 families have been describedwith deleterious mutations in the IL-12Rb1 gene (M1–M13, see Table 1 and Fig. 2, where M = mutation). Eightof these patients were reported to have missense muta-tions: R173P (M4), C198R (M7), R213W (M8) andQ214R (‘M9’). The rest have nucleotide substitutions ordeletions resulting in either splicing errors that causepremature truncation of the protein further downstream(M1, M3, M5, M6, M10, M11) or nonsense mutationsleading directly to a stop codon (M2, M12, M13).Interestingly, two patients (with different mutations) havebeen described in which aberrant splicing results in twotranscripts, one of which results in an in-frame deletion ofaa 137–193, thus potentially encoding a partially func-tional protein (M5, M6). However, no residual IL-12Rb1function or expression on the cell surface could bedetected in these patients (Elloumi-Zghal et al. 2002;Lichtenauer-Kaligis et al. 2003). All known mutations arerecessive.

Whereas these mutations are rare and can cause severeclinical disease, there are eight additional coding varia-tions in IL12RB1 known (V1–V8, see Table 1 and Fig. 2,

819

in which V = variation), four of which are foundfrequently: R156H (V2), Q214R (V3), M365T (V4) andG378R (V5). Q214R was earlier considered as a delete-rious mutation (‘M9’, see above), but now appears to be acommon polymorphism. Two variations, H438Y (V6) andG594E (V8), have each been found once in Japanesetuberculosis patients (Sakai et al. 2001), but not in any

other Japanese individuals. In addition, we have not foundG594E in 110 independent chromosomes of Caucasianindividuals (this report). A seventh variation, A525T(V7), was found heterozygously in three Japanese indi-viduals (Sakai et al. 2001). The eighth variation, S74R(V1), we found heterozygously in two Caucasian indi-viduals: a tuberculosis patient and a healthy Mantoux-

Fig. 2 Protein domains of IL-12Rb1 and genetic mutations andvariations. In the 662-aa IL-12Rb1 protein several functionaldomains can be distinguished. The N-terminal signal peptide (24 aa)is cleaved, giving a mature IL-12Rb1 protein of 638 aa. The 516-aaextracellular domain (ECD) contains the N-terminal cytokine-binding region (CBR, about 200 aa long) consisting of twofibronectin type III repeats harboring two Cys-Cys pairs (indicatedby black bars) and the [STGL]xWSxWS motif, which arecharacteristic gp130 cytokine receptor family signatures. Theremainder of the ECD consists of three additional fibronectintype III repeats, each about 100 aa in length. The ECD is followed

by a single 31-aa transmembrane domain and a short cytoplasmicportion (91 aa), containing box 1 (aa 577–584) and box 2 (aa 618–629) cytokine receptor motifs. Potential glycosylation sites aredepicted by black diamonds (aa 121, 329, 346, 352, 442, 456). Thedeleterious mutations (M) and variations (V) in the gene areindicated in the middle. The resulting changes in the protein areindicated on the right. Mutations that cause complex exon skippingresulting in frameshifts and eventually premature stops have beenindicated by premature stop. Mutations that in addition cause in-frame deletions have been indicated as complex pattern (for detailsand references see Table 1)

820

Tab

le1

Mut

atio

nsan

dva

riat

ions

repo

rted

inIL

12R

B1

(Mm

utat

ion,

Vva

riat

ion)

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orig

inal

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late

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und

tobe

apo

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din

5%(2

of44

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inde

pend

ent

chro

mos

omes

.V

2w

efo

und

in19

%(1

5of

80)

of

chro

mos

omes

.V3

we

foun

din

53%

(112

of21

2)of

chro

mos

omes

.V4

we

foun

din

47%

(34

of72

)of

chro

mos

omes

.V5

we

foun

din

56%

(61

of10

8)of

chro

mos

omes

.V8

we

did

not

find

in11

0ch

rom

osom

es.

All

indi

vidu

als

wer

eC

auca

sian

Mut

atio

nsin

IL12

RB

1(i

nN

M_0

0553

5)R

esul

ting

amin

oac

idch

ange

(fro

mm

ethi

onin

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efer

ence

M1

G12

8+5A

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ptic

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ltin

gin

seve

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Ell

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.20

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158T

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al.

1998

M3

531–

547:

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leti

onS

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ftat

137,

stop

at14

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2003

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2001

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at19

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02M

661

2–62

7:G

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GA

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137–

193)

Lic

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2003

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2001

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1998

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fram

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ftat

234,

stop

at26

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5+8

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ames

hift

at13

7,st

opat

144)

Lic

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11G

847+

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266)

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are

etal

.19

98a

M12

A97

7TK

305X

=st

opA

ltar

eet

al.

1998

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1190

TQ

376X

=st

opde

Jong

etal

.19

98

Var

iati

ons

ofun

know

nna

ture

(see

text

)

V1

C28

6GS

74R

Thi

sre

port

V2

G53

1AR

156H

Thi

sre

port

V6

C13

76T

H43

8YS

akai

etal

.20

01V

7G

1637

AA

525T

Sak

aiet

al.

2001

V8

G18

45A

G59

4ES

akai

etal

.20

01V

8G

1845

AG

594E

Thi

sre

port

Pol

ymor

phis

ms

V3

A70

5GQ

214R

Sak

aiet

al.

2001

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A70

5GQ

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Ell

oum

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3A

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4RT

his

repo

rtV

4T

1158

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aiet

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2001

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mi-

Zgh

alet

al.

2002

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repo

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1196

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378R

Sak

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al.

2001

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96C

G37

8RE

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mi-

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al.

2002

V5

G11

96C

G37

8RT

his

repo

rt

821

positive individual (from Arend et al. 2000). It is,however, not clear whether the last five coding variations(R156H, H438Y, G594E, A525T and S74R) should beconsidered polymorphisms or mutations, since they havenot yet been found homozygously in either a healthycontrol or a patient. This issue is further analyzed below.

Analyses of structural and functional effectsof variations in IL12RB1

In order to establish more precise relationships betweenstructure and function of the various domains in IL-12Rb1, and to gain more insight into the functional effectthat missense mutations and polymorphisms in IL12RB1may have on its function, we have used three approaches.First, we aligned the amino acid sequences of the IL-12Rb1 protein from different species to determine thedegree of conservation at the variable positions in theprotein. Second, we aligned IL-12Rb1 to gp130 andanalyzed reported gp130 b-sheets and helices, as well aspredicted sheets and helices in IL-12Rb1, to determinewhether any of the mutations and polymorphisms poten-tially alter the three dimensional structure of IL-12Rb1.Third, we examined the functions of the IL-12Rb1domains in which the mutations and polymorphisms werepresent. For several IL-12Rb1 domains whose functionhas not been determined, we inferred their function fromgp130 domains as determined by mutational experiments.

Cross-species alignment of IL-12Rb1

The complete amino acid sequence of the IL-12Rb1protein is available for only two species: Homo sapiens(P42701) and Mus musculus (Q60837). Using blastprograms (Altschul et al. 1990) to search anonymousESTs in dbEST, we were able to identify one EST fromMacaca mulatta (AF227552) and several sequences fromBos taurus (AW345812, BM366558, BF073865,AW653669) that were cloned in silico to generate one(incomplete) IL12RB1 transcript. In addition, one ESTfrom Rattus norvegicus (AF083328) was found that wasnot used in the alignment, since it was nearly identical tothe Mus musculus sequence, represented only a shortstretch of amino acids, and was not in a region in whichhuman mutations or polymorphisms have been identified.Figure 3 shows the alignments, with the mutations andpolymorphisms indicated.

Fig. 3 Cross-species alignment of IL-12Rb1 amino acid sequences.Alignment of the complete mature IL-12Rb1 amino acid sequencesof Homo sapiens (P42701) and Mus musculus (Q60837), partialamino acid sequence of Macaca mulatta (based on EST AF227552)and a partial sequence of Bos taurus (based on in silico clonedsequences from ESTs: AW345812, BM366558, BF073865,AW653669). The open triangles indicate polymorphisms, theclosed triangles indicate mutations, and triangles filled with greyare variations that could be either polymorphisms or mutations (seetext)

822

It is immediately obvious that each of the deleteriousmissense mutations described above (R173P, C198R andR213W) is in an amino acid that is identical for thedifferent species. In contrast, most of the polymorphismsin human IL12RB1 found homozygously in healthyindividuals are present in other species as functionalsubstitutions: e.g. Q214R is an R in both Mus musculusand Macaca mulatta; M365T is a T in Mus musculus andG378R is an R in M. musculus. Also, H438Y, which wasfound in a Japanese tuberculosis patient but not in healthyJapanese controls, is a Y in M. musculus. An exception tothis is R156H, a common variation that we found in 15out of 80 chromosomes in healthy controls, but onlyheterozygously, which is R in both humans and mice, butfor which no protein sequence is available in otherspecies.

The three rare variations were in conserved aminoacids: the A525T variation, is conserved in both humansand mice, but no protein sequence is available from otherspecies; the G594E variation is conserved in human,mouse and bovine sequences; and the S74R variation isalso conserved in all three species. In addition, G594Eand S74R are both substitutions of a small amino acid bya large polar amino acid. As mentioned previously, theselast three variations, A525T, G594E and S74R have notbeen found homozygously in healthy controls or patientsyet and could therefore represent either deleteriousmutations or harmless polymorphisms. Analysis of largergroups of patients and controls should clarify this issue.Based on this analysis, A525T, G594E and S74R maywell be deleterious mutations, whereas R156H andH438Y are most likely harmless polymorphisms.

Alignment of IL-12Rb1 to gp130 and effectsof genetic IL12RB1 variationson the three-dimensional structure of IL-12Rb1

We next determined the potential effect of mutations andother amino acid variations on the three-dimensionalstructure of the protein by aligning the amino acidsequences from IL-12Rb1 and gp130. In Fig. 4 we haveindicated the sheets and helices in gp130 as determinedby crystallography by Bravo and co-workers (1998), andthe IL-12Rb1 sheets and helices as predicted by Predict-Protein (Rost 1996). Bravo et al. determined the crystalstructure of the cytokine-binding region (CBR) (aa 100–303) of gp130. The two domains examined are homol-ogous to the two N-terminal fibronectin type III repeatsthat comprise the CBR in IL-12Rb1 (aa 25–216), andform a b-sandwich by connecting the two fibronectintype III repeats at an 80-degree angle. Since Bravo et al.only determined the sheets and helices as depicted in thefirst 200 aa of our alignment, we added sheets and helicesas predicted by PredictProtein to the remainder of theprotein in Fig. 4.

When we insert amino acids with known deleteriouseffects in the IL-12Rb1 protein, the three-dimensionalstructure of IL-12Rb1 is clearly affected since sheets or

Fig. 4 Alignment of IL-12Rb1 to gp130. Alignment of the humanamino acid sequences of IL-12Rb1 (P42701, aa 42–662 of the 662-aa protein) and gp130 (A36337, aa 124–736 of the 918-aa protein).The indicated sheets and helices for IL-12Rb1 are based onPredictProtein; for gp130 these are based on Bravo et al. (1998) forthe first 200 aa shown, the remainder are based on PredictProtein(Rost 1996). The open triangles indicate polymorphisms, the closedtriangles indicate mutations, and triangles filled with grey arevariations that could be either polymorphisms or mutations (seetext)

823

Tab

le2

Sum

mar

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anal

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foun

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moz

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sin

pati

ents

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foun

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notf

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inco

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ls,

CB

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toki

nebi

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gion

,n.

a.no

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alyz

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see

text

for

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ils

and

disc

ussi

on

Mis

sens

em

utat

ions

Var

iati

ons

ofun

know

nna

ture

Pol

ymor

phis

ms

R17

3PC

198R

R21

3W

S74

RR

156H

aH

438Y

A52

5Ta

G59

4EQ

214R

M36

5TG

378R

Pre

senc

ein

popu

lati

onP

P/C

cP

P/C

cP

P/C

cP

p/C

cP

p/C

cP

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helices according to PredictProtein are altered. TheR173P that is inside a sheet shortens that sheet by oneamino acid. The C198R makes the subsequent helix startwith one additional amino acid. The R213W, which is in ahelix, extends the helix by one more amino acid (Table 2).

In contrast, several of the variations did not affectsheets or helices in this analysis: in particular S74R andR156H, both in a sheet, and Q214R and M365T (whichare not in a sheet or helix). Instead, other variations doaffect predicted sheet or helical structures and thus mayaffect IL-12Rb1 structure. G378R reduces the subsequentsheet from TCSL to SL. H438Y, which is in a sheet,shortens that sheet by one amino acid. A525T makes bothadjacent sheets start with one additional amino acid.G594E has the strongest effect in that it shifts thepreceding sheet one amino acid forward, extends thefollowing sheet from WQ to ETWQWI, inserts a newhelix at QEE and shortens the last helix by the first aminoacid. Of interest is that three of these four variations,which are predicted to change the three-dimensionalstructure, have been found very rarely. G594E andH438Y have each been reported only once and onlyheterozygously (in tuberculosis patients), and A525T hasonly been found heterozygously in three individuals (seeabove) (Sakai et al. 2001). Based on the predictedstructural changes, these three variations are hypothesizedto be deleterious mutations. In the previous approach,based on evolutionary conservation, we concluded S74R,A525T and G594E to be deleterious, and H438Y as wellas R156H to be polymorphic variations. The first twoapproaches together indicate that A525T and G594E aredeleterious mutations, R156H is a polymorphism and thatS74R and H438Y can be either.

Variations in IL-12Rb1 domains with known function

The CBR in IL-12Rb1 consists of two fibronectin type IIIdomains, the first containing the two disulfide-bondforming Cys-Cys pairs and the second containing the[STGL]xWSxWS motif (Fig. 2). Only one variation,S74R, has so far been found in the first domain of theCBR, and is located between the two conserved Cys-Cyspairs, thus potentially interfering with cytokine binding(Fig. 2). Strikingly, all known deleterious missensemutations and two variations are located in the secondCBR domain. These mutations (R173P, C198R, R213W)thus affect the CBR and could interfere with IL-12 or IL-23 binding, thereby preventing signaling.

The R213W and R173P mutant proteins were unde-tectable at the cell surface and patients’ cells with thesemutations did not respond to exogenous IL-12 (Aksu et al.2001; Altare et al. 2001; Sakai et al. 2001). The C198Rmutant protein was also undetectable at the cell surface,but the cells from this patient were able to generate a lowresponse to IL-12 (Lichtenauer-Kaligis et al. 2003). BothC198R and R173P mutant proteins, however, could bedetected intracellularly (Lichtenauer-Kaligis et al. 2003;F.A.W. Verreck et al. unpublished data). (Unfortunately,

R213W has not yet been tested for intracellular expres-sion.) The intracellular presence of the mutant proteinssuggests that missense mutations in the second CBRdomain prevent transport to the cell surface, possibly byaltering the three-dimensional structure. It is unknownwhether this mechanism, or the potential interference withIL-12/IL-23 binding, or both, contribute(s) to the loss-of-function phenotype of these mutant proteins. Interesting-ly, the two variations that have been found in this domain,R156H and Q214R, are predicted not to affect any sheetsor helices (see above). One additional mutation (M6, seeTable 1) results in an in-frame deletion of exons 5 and 6in 50% of the mRNA molecules, potentially encoding amutant protein that selectively lacks half of the secondCBR domain (aa 137–193). The mutant protein was non-functional since there was no response to IL-12 stimula-tion. In addition, the mutant protein was detectableneither on the cell surface nor intracellularly in thepatients’ cells, indicating once more that this region iscrucial for the formation of a stable protein.

No mutations or polymorphisms have been reported inthe transmembrane domain. Comparison of the humanand mouse protein sequences (Fig. 3) shows that nine outof 31 aa in the TM domains differ, although mostsubstitutions are highly conserved. Mutations and varia-tions in the small TM domain will likely be found when alarger number of individuals have been analyzed.

Function of IL-12Rb1 domains inferredfrom mutational research in gp130

No naturally occurring mutations and polymorphisms ingp130 have been reported so far. However, in severalstudies mutant gp130 proteins were generated to study thefunctions of the individual domains. The results indicatethat D4, D5 and D6 (corresponding to the threemembrane-proximal fibronectin type III repeat domainsin IL-12Rb1) are involved in correct dimerization of thereceptor chains. D4 acts as a spacer, D5 allows dimer-ization between the two receptor chains and D6 ensurescorrect spacing and orientation of the cytoplasmicdomains of the receptors for signaling (Kurth et al.2000; Timmermann et al. 2002). No single amino acidchanges in these gp130 domains have been generated. Nodeleterious missense mutations have been reported so farin these IL-12Rb1 domains, although several amino acidvariations (M365T, G378R, H438Y, A525T) have beenfound. The fact that deleterious missense mutations inthese domains have not yet been found is not informative,since these may either be incompatible with life or,alternatively, only subtly (or not at all) impair thefunction of the protein.

Based on deletion mutants in the cytoplasmic region ofgp130, it was determined that box 1 (involved in Jakassociation) has a critical role in signal transduction(Murakami et al. 1991). In another study, using a singleamino acid change in the cytoplasmic region betweenbox 1 and box 2, it was found that a larger region than

825

box 1 is required for Jak-associated signal transduction(Haan et al. 2000). The IL-12Rb1 variation G594E, whichmay be either a deleterious mutation or a harmlesspolymorphism, resides within this larger Jak associationregion and can therefore have a similar effect. Thedomains of IL-12Rb1 and gp130 are schematicallyrepresented in Fig. 5.

IL12RB1 alternative splicing

An important natural source of variation in proteins isalternative splicing. When IL12RB1 was first identified,Northern blot analysis revealed two transcripts of 2.3 and3 kb, of which one appeared to lack the cytoplasmicdomain (Chua et al. 1994). A follow-up on this putativealternatively spliced product has not been published.Using blast searches (Altschul et al. 1990) on the humanEST database, several ESTs can be identified for thecurrently accepted transcript of IL12RB1 (NM_005535),in which transcription starts at a nucleotide positionfurther upstream, or extends further downstream. Thisaccounts for the two mentioned transcript sizes. None ofthe ESTs that appear to be genuine transcripts alter theprotein.

While sequencing IL12RB1 transcripts in patients(n=11) and controls (n=5), we found that half of theIL12RB1 transcripts lack the last 13 bp of exon 16. This

was due to alternative splicing (Fig. 6), resulting in a verysmall variation in the protein, in which the last four aminoacids, KAKM (lysine, alanine, lysine, methionine) arereplaced by DR (aspartate, arginine) (Chua et al. 1994).The functional significance of this variation is unclear,but we propose it differentially regulates protein stabilityin different cell types.

Discussion and concluding remarks

IL-12Rb1 is a key protein in the type-1 cytokine pathway,being an essential component of both IL-12R and IL-23R,which are both required for induction of optimal IFN-gproduction in response to infection with intracellularbacteria. In IL-12Rb1-deficient patients, poorly patho-genic mycobacteria and Salmonella species frequentlycause severe infections. Part of the genetic IL12RB1mutations found result in premature stops in the protein,explaining their deleterious effect. However, severaldeleterious (missense) mutations have been found thatalso result in complete IL-12Rb1 deficiency, whereasother amino acid variations do not affect its function.

Mutant proteins lacking cell surface expression

Since the structure-function relationship of IL-12Rb1 isnot well understood, we have used several approaches todetermine the impact of amino acid variations on IL-12Rb1 structure and function. Based on these analyses,we conclude that variations in the second domain of theCBR of IL-12Rb1 most likely affect the processing orfolding of the protein in such a way that the proteincannot be presented at the cell surface, presumably byaffecting structurally essential sheets or helices (as is thecase for R213W, R173P, C198R).

A protein quality control system in the endoplasmicreticulum (ER) prevents transport of mutant, misfolded,or incorrectly complexed proteins, and targets these for(rapid) degradation (Bross et al. 1999; Klausner and Sitia1990). Several transmembrane proteins are known inwhich missense mutations preclude expression of theprotein at the cell surface, but not intracellularly. In mostcases altered processing trapped the mutant proteins in theER (d’Addio et al. 2000; Iwashita et al. 1996; Sung et al.1993; Wojcik et al. 1998). Intracellular expression isdetected for the R173P and C198R mutant proteins aswell. Whether such IL-12Rb1 mutant proteins are local-ized in the ER or in a different subcellular organelle hasnot been determined. The R213W mutant protein wasundetectable in Western blotting (Sakai et al. 2001).Analysis of the subcellular localization in these and otherIL-12Rb1 missense mutants is needed to determine whereand at which stage of their assembly or maturation theseproteins get trapped.

Fig. 5 Schematic representation of IL-12Rb1 and gp130. Thegp130 chain contains a large extracellular segment comprised of sixdomains (indicated as D1–D6). The first domain is an Ig-likedomain (Ig), which is not present in IL-12Rb1. The other domains,also present in IL-12Rb1, are fibronectin type III repeats (FNIII), ofwhich the first two contain the cytokine receptor signature(WSxWS motif and two Cys-Cys pairs), together forming thecytokine-binding region (CBR). The last three FNIII domains arerequired for proper dimerization. The intracellular part of gp130contains, in addition to the box 1 and box 2 motifs also present inIL-12Rb1, a long signal-transducing region. Representation adapt-ed from (Kurth et al. 2000)

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Genetic variations of unknown nature

Of the five variations that have been found onlyheterozygously, S74R, R156H, G594E, H438Y andA525T, we tried to determine whether they are harmlesspolymorphisms or deleterious mutations. Based on theanalyses (summarized in Table 2) we conclude thatH438Y and R156H most likely represent polymorphisms,S74R can be either deleterious or harmless, and thatA525T and G594E most likely represent deleteriousmutations.

Only by identifying these variations homozygously, incontrol individuals or patients with defects in their type-1cytokine pathway, or by analyzing the function ofconstructs encoding the variant proteins in vitro can weverify the exact nature of these variations.

Acknowledgements This work was supported by grants from theNetherlands Organisation for Scientific Research (NWO/ZonMW)and the Netherlands Leprosy Foundation (NLR).

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