The stromal cell-derived factor-1/CXCL12 chemokine engages the CXCR4 and CXCR7 receptors and regulates homeostatic and pathologic processes, including organogenesis, leukocyte homeostasis, and tumorigenesis. Both receptors are widely expressed in mammalian cells, but how they cooperate to respond to CXCL12 is not well understood. Here, we show that CXCR7 per se does not trigger G(alphai) protein-dependent signaling, although energy transfer assays indicate that it constitutively interacts with G(alphai) proteins and undergoes CXCL12-mediated conformational changes. Moreover, when CXCR4 and CXCR7 are coexpressed, we show that receptor heterodimers form as efficiently as receptor homodimers, thus opening the possibility that CXCR4/CXCR7 heterodimer formation has consequences on CXCL12-mediated signals. Indeed, expression of CXCR7 induces conformational rearrangements within preassembled CXCR4/G(alphai) protein complexes and impairs CXCR4-promoted G(alphai)-protein activation and calcium responses. Varying CXCR7 expression levels and blocking CXCL12/CXCR7 interactions in primary T cells suggest that CXCR4/CXCR7 heterodimers form in primary lymphocytes and regulate CXCL12-promoted chemotaxis. Taken together, these results identify CXCR4/CXCR7 heterodimers as distinct functional units with novel properties, which can contribute to the functional plasticity of CXCL12.

One-third of the B400 nonodorant G protein-coupled receptors (GPCRs) are still orphans. Although a considerable number of these receptors are likely to transduce cellular signals in response to ligands that remain to be identified, they may also have ligand-independent functions. Several members of the GPCR family have been shown to modulate the function of other receptors through heterodimerization. We show that GPR50, an orphan GPCR, heterodimerizes constitutively and specifically with MT 1 and MT 2 melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT 2 did not modify MT 2 function, GPR50 abolished high-affinity agonist binding and G protein coupling to the MT 1 protomer engaged in the heterodimer. Deletion of the large C-terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT 1 without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT 1 . Pairing orphan GPCRs to potential heterodimerization partners might be of clinical importance and may become a general strategy to better understand the function of orphan GPCRs.

WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is an immune deficiency linked in many cases to heterozygous mutations causing truncations in the cytoplasmic tail of CXC chemokine receptor 4 (CXCR4). Leukocytes expressing truncated CXCR4 display enhanced responses to the receptor ligand CXCL12, including chemotaxis, which likely impair their trafficking and contribute to the immunohematologic clinical manifestations of the syndrome. CXCR4 desensitization and endocytosis are dependent on ␤-arrestin (␤arr) recruitment to the cytoplasmic tail, so that the truncated CXCR4 are refractory to these processes and so have enhanced G protein-dependent signaling. Here, we show that the augmented responsiveness of WHIM leukocytes is also accounted for by enhanced ␤arr2-dependent signaling downstream of the truncated CXCR4 receptor. Indeed, the WHIM-associated receptor CXCR4 1013 maintains association with ␤arr2 and triggers augmented and prolonged ␤arr2-dependent signaling, as revealed by ERK1/2 phosphorylation kinetics. Evidence is also provided that CXCR4 1013 -mediated chemotaxis critically requires ␤arr2, and disrupting the SHSK motif in the third intracellular loop of CXCR4 1013 abrogates ␤arr2-mediated signaling, but not coupling to G proteins, and normalizes chemotaxis. We also demonstrate that CXCR4 1013 spontaneously forms heterodimers with wild-type CXCR4. Accordingly, we propose a model where enhanced functional interactions between ␤arr2 and receptor dimers account for the altered responsiveness of WHIM leukocytes to CXCL12. (Blood. 2008;112:34-44) IntroductionThe G-protein-coupled receptor (GPCR) CXC chemokine receptor 4 (CXCR4) and its ligand, the stromal cell-derived factor-1 (CXCL12/SDF-1), regulate leukocyte hematopoiesis and trafficking. 1 They initiate signal transduction by activating heterotrimeric G␣␤␥-proteins, which then act on effectors to trigger downstream cellular responses. 2 CXCL12 also elicits processes causing receptor desensitization, which results in the uncoupling from G-proteins and involves phosphorylation of the CXCR4 cytoplasmic tail (C-tail) by protein kinase C and GPCR kinases (GRKs) and interaction of the phosphorylated receptor with ␤-arrestins (␤arrs). [3][4][5] ␤arrs then target desensitized CXCR4 to clathrin-coated pits for endocytosis. ␤arrs also link GPCRs to the stimulation of additional signaling molecules, including members of the mitogen-activated protein kinase (MAPK) family. 6 Studies on CXCR4 have demonstrated that ␤-arrestin2 (␤arr2) strengthens activation of the p38 and extracellular signal-regulated kinase (ERK) MAPKs and CXCL12-induced chemotaxis. 5,7,8 WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome (WS) is a rare immunodeficiency disease linked to CXCR4 dysfunctions and is characterized by warts, recurrent bacterial infections, hypogammaglobulinemia, lymphopenia, and myelokathexis, a severe neutropenia with abnormal retention of mature neutrophils in the bone marrow (BM). 9,10 WS, most often inherited as an a...