Chemokine receptor dimerization: two are better than one

612

Opinion

27 Ignatowicz, L. et al. (1996) The repertoire of T cells shaped by a single MHC/peptide ligand. Cell 84, 521–529 28 Miyazaki, T. et al. (1996) Mice lacking H2-M complexes, enigmatic elements of the MHC class II peptide-loading pathway. Cell 84, 531–541 29 Zerrahn, J. et al. (1997) The MHC reactivity of the T-cell repertoire prior to positive and negative selection. Cell 88, 627–636 30 Clark, S.P. et al. (1995) Comparison of human and mouse T-cell receptor variable-gene-segment subfamilies. Immunogenetics 42, 531–540 31 Chen, F. et al. (2001) Differential transcriptional regulation of individual TCR Vβ segments before gene rearrangement. J. Immunol. 166, 1771–1780 32 Aude-Garcia, C. et al. (2001) Preferential ADV-AJ association during recombination in the mouse

TRENDS in Immunology Vol.22 No.11 November 2001

33

34

35

36

37

T-cell receptor αδ locus. Immunogenetics 52, 224–230 McMurry, M.T. and Krangel, M.S. (2000) A role for histone acetylation in the developmental regulation of VDJ recombination. Science 287, 495–498 Sleckman, B.P. et al. (1998) Accessibility control of variable region gene assembly during T-cell development. Immunol. Rev. 165, 121–130 Davis, M.M. and Bjorkman, P.J. (1988) T-cell antigen-receptor genes and T-cell recognition. Nature 334, 395–402 Blom, B. et al. (1999) TCR gene rearrangements and expression of the pre-T-cell-receptor complex during human T-cell differentiation. Blood 93, 3033–3043 Hassanin, A. et al. (2000) Evolution of the recombination signal sequences in the Ig heavychain variable-region locus of mammals. Proc. Natl. Acad. Sci. U. S. A. 97, 11415–11420

Chemokine receptor dimerization: two are better than one José Miguel Rodríguez-Frade, Mario Mellado and Carlos Martínez-A. The chemokines participate in an exceptional range of physiological and pathological processes, including the control of lymphocyte trafficking, tumor growth, wound healing, allograft rejection, regulation of T-cell differentiation, asthma, infection with HIV and atherosclerosis. This vast array of activities is triggered by the interaction of nearly 50 different chemokines with a relatively modest number of 20 G-protein-coupled receptors. The asymmetry between the number of receptors and ligands suggests an underlying, shared control mechanism activated at a very early stage of the response. One of the first events triggered by the binding of chemokines is the homo- and heterodimerization of their receptors; here, we outline these events and their consequences in chemokine signaling.

José Miguel Rodríguez-Frade Mario Mellado Carlos Martínez-A.* Dept of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, UAM Campus de Cantoblanco, E-28049 Madrid, Spain. *e-mail: cmartineza@ cnb.uam.es

Patrolling an organism to identify ‘aliens’ or uncontrolled cells is a fundamental attribute of cells of the immune system; this function is triggered mainly by chemoattractants. In mammals, an essential set of these molecules is a class of chemoattractant cytokines, the chemokines, which exert their activity by binding to members of the large family of G-protein-coupled receptors (GPCRs). Following the description of the first chemokine, CXCL4 [platelet factor 4 (PF4)], many new members have been discovered, forming a family of secreted, low-molecular-weight proteins (of approximately 70 amino acids)1, involved not only in the migration and activation of leukocytes, but also, in http://immunology.trends.com

38 Davis, M.M. et al. (1984) A murine T-cell-receptor gene complex: isolation, structure and rearrangement. Immunol. Rev. 81, 235–258 39 Rowen, L. et al. (1996) The complete 685 kB DNA sequence of the human β T-cell-receptor locus. Science 272, 1755–1762 40 Arstila, T.P. et al. (1999) A direct estimate of the human αβ T-cell-receptor diversity. Science 286, 958–961 41 Hesse, J.E. et al. (1989) V(D)J recombination: a functional definition of the joining signals. Genes Dev. 3, 1053–1061 42 Thompson, J.D. et al. (1997) The CLUSTAL-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882 43 Saitou, N. and Nei, M. (1987) The neighborjoining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425

inflammation, hematopoiesis, angiogenesis, tumor rejection, T helper 1 (Th1)- versus Th2-cell polarization and responses, and the control of infection with HIV-1 (Refs 2–6). Depending on their physiological features, which include the conditions and location of production, as well as the cellular distribution of their receptors, chemokines are classified as being inflammatory and inducible, or homeostatic and constitutive. This functional classification replaces the original organization, based on criteria of structure and chromosomal location, which designated four groups (CXC, CC, C and CX3C) based on the relative positions of the N-terminal cysteine residues found in the majority of chemokines1,7,8.

‘...these data support a model that indicates an early, crucial role for JAKs in chemokine signaling...’ Chemokines exert their effects by interaction with seven-transmembrane domain GPCRs in the target-cell membrane. All of these receptors have an approximate molecular weight of 40 kDa. They are classified into four families – CCR, CXCR, CR and CX3CR – according to the type of chemokine to which they bind. The extracellular domain consists of an N-terminus and three extracellular loops, which act in concert to bind to the chemokine ligand; the intracellular region is composed of three loops and the C-terminus, which collaborate to transduce the chemokine signal and regulate expression of the receptor. Similar to many other seven-transmembrane domain receptors, the chemokine receptors have several conserved structural features, such as the DRYLAIV amino acid sequence in the second intracellular loop. The relevance of this motif to chemokine signaling is discussed later9.

1471-4906/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1471-4906(01)02036-1

Opinion

Fig. 1. Chemokines trigger the JAK–STAT pathway. (a) The binding of a chemokine to its corresponding receptor exposes the tyrosine residue in the DRYLAIV motif of the intracellular domain of the receptor. (b) This allows access of Janus kinases (JAKs), which activate the receptor by tyrosine phosphorylation [indicated by a change in the color of the tyrosine residue (Y) from red to green]. (c) The activation of JAK (green to blue color change) induces the translocation of signal transducer and activator of transcription (STAT) and its association with the receptor complex. As a result of the ligandpromoted conformational changes in the receptor and the association of JAK, the G-proteinbinding epitope of the receptor is exposed, allowing the activation of G protein.

TRENDS in Immunology Vol.22 No.11 November 2001

(a)

613

Receptor

JAK

STAT

CXCR4

1 2 3

1 2 3 4 5b 6

CCR2

1 2 3

1 2 3 4 5b 6

CCR5

1 2 3

1 2 3 4 5b 6

TRENDS in Immunology

Y

JAK

(b)

JAK

Y

G protein STAT

(c)

JAK G protein

Y STAT

TRENDS in Immunology

Chemokines trigger tyrosine phosphorylation of their receptor and JAK–STAT activation

The classical view of signaling by receptors for chemoattractants involves activation of the G-protein pathway. Indeed, the majority of the responses to chemokines is inhibited by pertussis toxin (PTx), indicating that a Gi protein is involved in signal transduction10, and the association of Gαi with several chemokine receptors (e.g. CXCR1, CXCR4, CCR2 and CCR5) has been described11–14. As a consequence of this association, chemokines trigger the agonist-dependent inhibition of adenylyl cyclase and mobilization of intracellular calcium. However, PTx does not block the calcium response in some instances, and other, PTx-insensitive http://immunology.trends.com

Fig. 2. A summary of the JAK–STAT family members implicated in signaling by chemokine receptors. Janus kinase (JAK) and signal transducer and activator of transcription (STAT) molecules indicated in black are activated by the particular receptor; those in red are not activated by the receptor; and for those in blue, receptor-mediated activation has not been determined yet.

G proteins can bind to chemokine receptors; this indicates that non-Gi proteins might be involved in chemokine signaling also15,16. It has been proposed that the chemokines – specifically CCL5 [regulated on activation, normal T-cell expressed and secreted (RANTES)] acting through the CCR5 receptor – activate the expression of genes by the phosphorylation and nuclear translocation of signal transducer and activator of transcription (STAT) also17. This idea is complemented by the observations that chemokine receptors are tyrosine phosphorylated and several members of the Janus kinase (JAK) family associate with activated receptors independent of the association of Gi (Ref. 18). Such a pathway was first described for CCR2, the CCL2 [monocyte chemoattractant protein 1 (MCP-1)] receptor13; in this case, the activation of JAK2 was required for the coupling of G protein to the receptor (Fig. 1). Following binding of CCL2 to CCR2, the Y139 residue in the conserved chemokine receptor DRYLAIV motif is probably the primary target for JAK2-mediated phosphorylation of the CCR2 receptor. Mutation of this tyrosine to a phenylalanine (CCR2bY139F) results in a ‘dead’ receptor that is unable to recruit or activate JAK2, thus impairing the activation of Gi. The similarities between chemokine receptors, including conservation of the DRYLAIV motif, were considered to be an indication that activation of the JAK–STAT pathway is not exclusive to the triggering of CCR2, but might also be induced by other chemokine receptors, including other members of the CCR and CXCR families. This is the case for CCR5 and CXCR4, both of which activate distinct JAK–STAT proteins (Fig. 2)12,14,19. In CCR5-expressing HEK 293 cells stimulated with CCL5, CCR5 is tyrosine phosphorylated rapidly, and JAK1, but not JAK2 or JAK3, associates with the receptor. This promotes the association of STAT-5b with the receptor, as well as its tyrosine phosphorylation and activation14. CXCL12 [stromal-cell-derived factor 1α (SDF-1α)] promotes the rapid activation of JAK1 and JAK2 and their

Opinion

614

TRENDS in Immunology Vol.22 No.11 November 2001

(a)

DSS

(b) Agonist anti-CCR2 mAb

Anti-Fab Ab Anti-CCR2 Fabs

Signal

No signal

Anti-CCR2 Fabs

Signal

(c)

Y

Y

Signal

F

F

No signal

F

Y

No signal TRENDS in Immunology

Fig. 3. Chemokines promote receptor dimerization. Schematic representation of the three experimental strategies used to demonstrate chemokine-mediated receptor dimerization. (a) Two different epitope-tagged receptors are used; the presence of one (green tag) is evaluated in immunoprecipitations of the other (blue tag) (left panel). Similar experiments were performed using disuccinimidyl suberate (DSS) to crosslink the chemokine receptors after binding to ligand (right panel). (b) Signaling requires at least two chemokine receptors. The agonist activity of an anti-CCchemokine receptor 2 (anti-CCR2) monoclonal antibody (mAb) is lost when its fragments of antigen binding (Fabs) are used; activity is restored when the Fabs are crosslinked by an anti-Fab Ab. (c) A receptor with a mutation (Y to F) in the DRYLAIV motif does not trigger signaling after binding to its ligand; when cotransfected with the wild-type receptor, the mutant receptor has a dominant-negative effect, impeding ligand-induced activation.

association with CXCR4, followed by the phosphorylation of STAT-1, -2, -3 and -5b, and their activation and association with CXCR4 (Fig. 2)12,20. Taken together, these data support a model that indicates an early, crucial role for JAKs in chemokine signaling (Figs 1,2), and concur with several recent reports of the activation of the JAK–STAT pathway by other members of the GPCR family, including the CAR1 chemoattractant receptor in Dictyostelium, and the receptors for angiotensin II, α-melatoninstimulating hormone, serotonin and thyrotropinstimulating hormone in mammalian cells21–23. For all of these GPCRs, activation of the JAK–STAT pathway appears to be independent of G-protein signaling.

homo- or hetero-dimerization of their receptors, which then triggers the transphosphorylation and activation of JAKs (Ref. 24). The transphosphorylation and activation of JAKs might occur also through dimerization or oligomerization of the chemokine receptors, as indicated for CCR2 by several experimental approaches, including the use of tagged receptors, agonist monoclonal antibodies (mAbs) and mutant ‘dead’ receptors (Fig. 3)25. Using two pools of CCR2 tagged with two distinct epitopes, one tagged receptor is detected in immunoprecipitates of the other only after receptor activation mediated by the binding of ligand. In similar experiments using bifunctional reagents to crosslink the chemokine receptors after ligand binding, we found both monomers and species of higher molecular weight corresponding to dimers (Fig. 3a). Also, we have derived an agonist mAb for CCR2, which triggers Ca2+ mobilization and cell migration. Fragments of antigen binding (Fabs) of this mAb are unable to trigger biological responses, but the responses are recovered following the crosslinking of Fabs (Fig. 3b). Finally, although the CCR2bY139F mutant described previously is expressed correctly on the cell surface and binds to CCL2, binding to its ligand does not trigger a biological response. The coexpression of mutant CCR2bY139F and wild-type CCR2 abrogates the biological response of the latter to CCL2 (Fig. 3c). Biochemical analysis of the complex formed on the cell surface shows that both receptors undergo dimerization in the presence of ligand; however, the wild-type–mutant receptor complex associates with neither JAK–STAT nor G protein. These approaches illustrate the dimerization of chemokine receptors during chemokine signaling and allow us to infer the significance of the DRYLAIV motif for the recruitment of JAK to the receptor complex. Similar observations have been made for other CCRs and for CXCRs, suggesting that homodimerization is a mechanism common to chemokine receptors when activated by their respective ligands18. Although the importance of the dimerization of GPCRs in signaling has only attracted the attention of the scientific community recently, it was first suggested 25 years ago and corroborative evidence has accumulated over this period. The results of studies similar to those used to demonstrate dimerization for the tyrosine kinase receptors26,27, the techniques described above25,28–31, as well as the newer biophysical, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) approaches32–34, lead to the conclusion that dimerization might be responsible for the biological responses induced by chemokine receptors.

Chemokines induce receptor dimerization

Activation of the JAK–STAT pathway was first identified during signaling by members of the cytokine receptor family. Binding to cytokines induces http://immunology.trends.com

Chemokines induce receptor heterodimerization

In a simple ‘one chemokine–one receptor’ model, a chemokine interacts with its receptor to induce the

Opinion

TRENDS in Immunology Vol.22 No.11 November 2001

615

Migration

Adhesion JAK–STAT

PTx

Gαq/11

Gαi

Reduced threshold

G protein

Time

Surface receptor expression (%)

Surface receptor expression (%)

PTx-sensitive

Time

No internalization No desensitization

Internalization Desensitization PI 3-K activity

PI 3-K activity

PI 3-K

Time

Time TRENDS in Immunology

Fig. 4. Chemokine receptor heterodimerization triggers distinct signaling events. The simultaneous presence of two different chemokines induces the formation of chemokine receptor heterodimers. In contrast to homodimers, the heterodimeric complex promotes specific recruitment of Gαq/11, is not internalized or desensitized to a second stimulus, has distinct phosphoinositide 3-kinase (PI 3-K) activation kinetics and preferentially activates cell adhesion rather than migration. Abbreviation: PTx, pertussis toxin.

formation of or stabilize receptor dimers or oligomers, permitting activation of the JAK–STAT pathway and G-protein coupling. We have observed that the simultaneous presence of CCL2 and CCL5 induces the formation of CCR2–CCR5 heterodimers, as would be predicted by the considerable sequence similarity between these two chemokine receptors35. The heterodimeric receptor complex has unique features, including a reduction in the threshold concentration of chemokine required to induce PTx-resistant responses; this suggests the activation of biochemical pathways different from those triggered by homodimers. The heterodimeric complex promotes the specific recruitment of Gq/11, which explains the PTx-resistant Ca2+ flux. It has distinct phosphoinositide 3-kinase (PI 3-K) activation kinetics, and preferentially activates cell adhesion, in contrast to the cell migration triggered by homodimers. Finally, the heterodimeric complex is http://immunology.trends.com

not internalized or desensitized to a second stimulus (Fig. 4)35. The differences between the biochemical pathways activated by homo- and hetero-dimers might have functional relevance. In inflammatory processes, many soluble mediators are present and, normally, a cell expresses several different chemokine receptors; thus, the possibility that different, specific chemokine pairs promote receptor heterodimerization adds new complexity to the physiology of chemokines. The availability of chemokines dictates the formation of signaling-domain complexes, thus regulating receptor sensitivity. The chemokines are produced within specific tissues where they are immobilized by low-affinity binding to heparinbearing proteoglycans on the vascular endothelial barrier; this permits effective presentation of chemokines to ‘rolling’ leukocytes in the circulation36. Therefore, variations in the availability of these presentation molecules or chemokines would affect dramatically the ability of a ligand to trigger the formation of homo- or hetero-dimers. At low concentrations of chemokines, receptor heterodimerization would be favored, cell adhesion would be triggered and therefore, leukocyte ‘rolling’ would be stopped, promoting the transvasation and migration of circulating cells into the tissues (Fig. 5).

Opinion

616

TRENDS in Immunology Vol.22 No.11 November 2001

Heterodimer Adhesion Homodimer

Key: Selectin Adhesion molecule

Migration

Proteoglycan Chemokines Chemokine receptors Integrin

TRENDS in Immunology

Fig. 5. The physiological role of dimerization of chemokine receptors. Leukocytes roll along the blood vessel endothelium; exposure to low concentrations of chemokines causes the formation of chemokine receptor heterodimers and the cell adheres to the endothelium. In the vicinity of the chemokine source, where concentrations of chemokines are high, homodimerization takes place. Cell migration towards this source would result in an inflammatory response or the accumulation of leukocytes in lymphoid organs. The main molecules involved in leukocyte extravasation are indicated.

Acknowledgements We thank A. Martín de Ana, S.F. Soriano and P. Hernanz for much of the work that contributed to this review, and C. Mark for editorial assistance. Our work was partially supported by grants from the CICyT, European Union/FEDER and the Comunidad Autónoma de Madrid. The Department of Immunology and Oncology was founded and is supported by the Spanish National Research Council (CSIC) and by Pharmacia Corporation.

Homodimerization would take place in the vicinity of the source of the chemokine; cell migration towards this source would result in an inflammatory response or the accumulation of leukocytes in lymphoid organs. A selective advantage of the homo- or heterodimerization of receptors lies in the increased sensitivity and specificity of the system37,38, such that the range of responses to chemokines is increased by the spread of activity through a receptor array, as suggested by the abundance and variety of chemokine receptors expressed on cell surfaces. Chemokine receptor activation: a whole new ballgame

Binding to chemokines induces sequential conformational changes in a receptor that lead finally to the formation of a signaling complex, the ‘chemosome’, responsible for the activation of chemokine-related signaling events. Binding to ligand induces changes in the conformation of the receptor that expose the residues required for dimerization. In the case of other GPCRs, for example the β-adrenergic receptor, these residues have been characterized and are located in transmembrane domain VI (Ref. 39), where they form a putative glycine- and leucine-rich dimerization motif. Similar motifs are found in several chemokine receptors; in http://immunology.trends.com

the case of CCR2 and CCR5, the motif is located between amino acids 43–70 in transmembrane domain I. In CCR2, a substitution of the valine at position 64 enables CCR2V64I to heterodimerize with CXCR4, whereas wild-type CCR2 is unable to do so. This might be related to the delayed progression to AIDS in HIV-1-infected individuals bearing this polymorphism40,41. As a consequence of binding to ligand, the tyrosine residue in the DRYLAIV motif is exposed within the cytoplasm42, allowing access of JAKs to the receptor, which are then tyrosine phosphorylated13. During cytokine-receptor-mediated activation of JAKs, JAK molecules associate constitutively with the receptor through a consensus motif43. By contrast, for the chemokine receptors, association with JAKs is promoted by the binding of ligand to the receptor, through a motif that has not been identified as yet. The association of a JAK with the chemokine receptor, but not its dissociation, occurs in the presence of PTx, but Gαi does not associate with the receptor when cells are pretreated with a JAKspecific inhibitor. This indicates that the association of Gαi with the receptor is JAK-dependent, and suggests a role for Gαi in fine-tuning the JAK–STAT pathway13.

…homodimerization is a ‘… mechanism common to chemokine receptors when activated by their respective ligands.’ Conclusion: are two receptors better than one?

Oligodimerization is an extremely efficient way to increase specificity and sensitivity because the assembly of different combinations of receptors and signaling molecules can generate variability in receptor subtypes, thus amplifying the capacity for a variable response44. This process operates in antigenreceptor signaling, cytokine responses, Fas-mediated cell death, the regulation of gene transcription, and in so many other cases that it clearly constitutes a major mechanism of cellular responses. Ligands can induce the dimerization of receptors in a number of ways45. Some extracellular ligands are themselves dimers, possessing two receptor-binding surfaces; other, monomeric ligands use two different surface sites to contact two receptor molecules. In other cases, monomeric ligands associate with heparin sulfate proteoglycans to form a multivalent complex, which in turn, binds to two or more receptors46. Despite advances in the identification and characterization of chemokine-receptor dimerization, the stoichiometry of the ligand–receptor complex and the dynamic orientation of its components remain to be defined. In any case, dimerization has only been characterized in in vitro systems and its full significance needs to be demonstrated using in vivo models. However, cells

Opinion

TRENDS in Immunology Vol.22 No.11 November 2001

derived from CCR5∆32 homozygous individuals fail to undergo the responses associated with CCR2–CCR5 receptor heterodimerization35. An implication of receptor clustering is that the activity of one receptor would influence that of its neighbors, permitting a ligand to act in trans on a receptor for which it is not specific. Thus, the bound ligand induces changes in the signaling activity of the receptor that are propagated to a large number of neighboring receptors, boosting the effect of a single binding event. In other words, chemokine binding triggers receptor clustering that adapts to external stimuli. The correlation between the differential References 1 Rossi, D. and Zlotnik, A. (2000) The biology of chemokines and their receptors. Annu. Rev. Immunol. 18, 217–242 2 Murdoch, C. and Finn, A. (2000) Chemokine receptors and their role in inflammation and infectious diseases. Blood 95, 3032–3043 3 Sallusto, F. et al. (2000) The role of chemokine receptors in primary, effector and memory immune responses. Annu. Rev. Immunol. 18, 593–620 4 Berger, E.A. et al. (1999) Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism and disease. Annu. Rev. Immunol. 17, 657–700 5 Gerard, C. and Rollins, B.J. (2001) Chemokines and disease. Nat. Immunol. 2, 108–115 6 Baggiolini, M. and Loetscher, P. (2000) Chemokines in inflammation and immunity. Immunol. Today 21, 418–420 7 Mackay, C.R. (2001) Chemokines: immunology’s high impact factors. Nat. Immunol. 2, 95–101 8 Rollins, B.J. (1997) Chemokines. Blood 90, 909–928 9 Murphy, P.M. (1994) The molecular biology of leukocyte chemoattractant receptors. Annu. Rev. Immunol. 12, 593–633 10 Thelen, M. (2001) Dancing to the tune of chemokines. Nat. Immunol. 2, 129–134 11 Damaj, B.B. et al. (1996) Identification of G-protein-binding sites of the human interleukin-8 receptors by functional mapping of the intracellular loops. FASEB J. 12, 1426–1434 12 Vila-Coro, A.J. et al. (1999) The chemokine SDF-1α triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway. FASEB J. 13, 1699–1710 13 Mellado, M. et al. (1998) The chemokine MCP-1 triggers JAK2 kinase activation and tyrosine phosphorylation of the CCR2b receptor. J. Immunol. 161, 805–813 14 Rodríguez-Frade, J.M. et al. (1999) Similarities and differences in RANTES- and (AOP)RANTES-triggered signals: implications for chemotaxis. J. Cell Biol. 144, 755–765 15 Arai, H. and Charo, I.F. (1996) Differential regulation of G-protein-mediated signaling by chemokine receptors. J. Biol. Chem. 271, 21814–21819 16 Al-Aoukaty, A. et al. (1996) Differential coupling of CC-chemokine receptors to multiple heterotrimeric G proteins in human interleukin-2-activated natural killer cells. Blood 87, 4255–4260 17 Wong, M. and Fish, E.N. (1998) RANTES and MIP-1α activate STATs in T cells. J. Biol. Chem. 273, 309–314

http://immunology.trends.com

617

induction of expression of chemokines and receptor configuration is still unknown, but the availability of chemokines might regulate receptor sensitivity by inducing the formation of diverse signaling complexes. Although new pathways activated by chemokine receptors are being identified regularly, many questions remain unanswered. The resolution of structure for a receptor and its ligand–receptor complex will aid in obtaining rapid answers to these questions. Because the chemokines are involved, one way or another, in a broad array of pathologies, this knowledge will have immediate impact on the design of a host of new drugs.

18 Mellado, M. et al. (2001) Chemokine signaling and functional responses: the role of receptor dimerization and TK pathway activation. Annu. Rev. Immunol. 19, 397–421 19 Zang, X.F. et al. (2001) Janus kinase 2 is involved in stromal-cell-derived-factor-1α-induced tyrosine phosphorylation of focal adhesion proteins and migration of hematopoietic progenitor cells. Blood 97, 3342–3348 20 Wong, M. et al. (2001) RANTES activates JAK2 and JAK3 to regulate engagement of multiple signaling pathways in T cells. J. Biol. Chem. 276, 11427–11431 21 Araki, T. et al. (1998) Developmentally and spatially regulated activation of a Dictyostelium STAT protein by a serpentine receptor. EMBO J. 17, 4018–4028 22 Ali, M.S. et al. (2000) JAK2 acts both as STAT1 kinase and as a molecular bridge linking STAT1 to the angiotensin II AT1 receptor. J. Biol. Chem. 275, 15586–15593 23 Park, E.S. et al. (2000) Involvement of JAK/STAT (Janus kinase/signal transducer and activator of transcription) in the thyrotropin signaling pathway. Mol. Endocrinol. 14, 662–670 24 Liu, K.D. et al. (1998) JAK/STAT signalling by cytokine receptors. Curr. Opin. Immunol. 10, 271–278 25 Rodríguez-Frade, J.M. et al. (1999) The chemokine monocyte chemoattractant protein 1 induces functional responses through dimerization of its receptor CCR2. Proc. Natl. Acad. Sci. U. S. A. 96, 3628–3633 26 Odaka, M. et al. (1997) Ligand-binding enhances the affinity of dimerization of the extracellular domain of the epidermal growth factor receptor. J. Biochem. 122, 116–121 27 Cunningham, S.A. et al. (1999) Characterization of vascular endothelial cell growth factor interactions with the kinase-insert-domaincontaining receptor tyrosine kinase. A real-time kinetic study. J. Biol. Chem. 274, 18421–18427 28 Salahpour, A. et al. (2000) Functional significance of oligomerization of G-protein-coupled receptors. Trends Endocrinol. Metab. 11, 163–168 29 Maggio, R. et al. (1993) Coexpression studies with mutant muscarinic/adrenergic receptors provide evidence for intermolecular ‘cross-talk’ between G-protein-linked receptors. Proc. Natl. Acad. Sci. U. S. A. 90, 3103–3107 30 Monnot, C. et al. (1996) Polar residues in the transmembrane domains of the type 1 angiotensin II receptor are required for binding and coupling. Reconstitution of the binding site by coexpression of two deficient mutants. J. Biol. Chem. 271, 1507–1513

31 Hebert, T.E. et al. (1998) Functional rescue of a constitutively desensitized β2AR through receptor dimerization. Biochem. J. 330, 287–293 32 Janetopoulos, C. et al. (2001) Receptor-mediated activation of heterotrimeric G proteins in living cells. Science 291, 2408–2411 33 Overton, M.C. and Blumer, K.J. (2000) G-proteincoupled receptors function as oligomers in vivo. Curr. Biol. 10, 341–344 34 Angers, S. et al. (2000) Detection of β2-adrenergicreceptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. U. S. A. 97, 3684–3689 35 Mellado, M. et al. (2001) Chemokine receptor homo- or hetero-dimerization activates distinct signaling pathways. EMBO J. 20, 2497–2507 36 Middleton, J. et al. (1997) Transcytosis and surface presentation of IL-8 by venular endothelial cells. Cell 91, 385–395 37 Kuner, R. et al. (1999) Role of heteromer formation in GABAB receptor function. Science 283, 74–77 38 Jordan, B.A. and Devi, L.A. (1999) G-proteincoupled receptor heterodimerization modulates receptor function. Nature 399, 697–700 39 Hebert, T.E. et al. (1996) A peptide derived from a β2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J. Biol. Chem. 271, 6384–6392 40 Mellado, M. et al. (1999) Chemokine control of HIV-1 infection. Nature 400, 273–274 41 Smith, M.W. et al. (1997) Contrasting genetic influence of CCR2 and CCR5 variants on HIV-1 infection and disease progression. Hemophilia Growth and Development Study (HGDS), Multicenter AIDS Cohort Study (MACS), Multicenter Hemophilia Cohort Study (MHCS), San Francisco City Cohort (SFCC), ALIVE Study. Science 277, 959–965 42 Bockaert, J. and Pin, J.P. (1999) Molecular tinkering of G-protein-coupled receptors: an evolutionary success. EMBO J. 18, 1723–1729 43 Bach, E.A. et al. (1997) The IFN-γ receptor: a paradigm for cytokine receptor signaling. Annu. Rev. Immunol. 15, 563–591 44 Klemm, J.D. et al. (1998) Dimerization as a regulatory mechanism in signal transduction. Annu. Rev. Immunol. 16, 569–592 45 Heldin, C.H. (1995) Dimerization of cell-surface receptors in signal transduction. Cell 80, 213–223 46 Luster, A.D. et al. (1995) The IP-10 chemokine binds to a specific cell-surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation. J. Exp. Med. 182, 219–231