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 Gαi protein–dependent signaling, although energy transfer assays indicate that it constitutively interacts with Gαi 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/Gαi protein complexes and impairs CXCR4-promoted Gαi-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 ∼400 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 MT1 and MT2 melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT2 did not modify MT2 function, GPR50 abolished high‐affinity agonist binding and G protein coupling to the MT1 protomer engaged in the heterodimer. Deletion of the large C‐terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT1 without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT1. 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.

Heterodimerization has been documented for several members of the G protein-coupled receptor (GPCR) superfamily, including the closely related MT(1) and MT(2) melatonin receptors. However, the relative abundance of hetero-versus homodimers and the specific properties, which can be attributed to each form, are difficult to determine. Using a bioluminescence resonance energy transfer (BRET) donor saturation assay, we show that half-maximal MT(1)/MT(2) heterodimer formation is reached for expression levels as low as approximately 4000 receptors per cell. The relative propensity of MT(1) homodimer and MT(1)/MT(2) heterodimer formation are similar, whereas that for the MT(2) homodimer formation is 3- to 4-fold lower. These data indicate that both the relative expression level of each receptor isoform and the affinities between monomers may determine the actual proportion of homo- and heterodimers. The specific interaction of ligands with the MT(1)/MT(2) heterodimer was studied using a BRET-based assay as a readout for the conformational changes of the heterodimer. An MT(1)/MT(2) heterodimer-specific profile and ligands selective for the MT(1)/MT(2) heterodimer compared with the MT(2) homodimer could be identified. Classic radioligand binding and BRET studies suggest that heterodimers contain two functional ligand binding sites that maintain their respective selectivity for MT(1) and MT(2) ligands. Occupation of either binding site is sufficient to induce a conformational change within the heterodimer. Taken together, these results show that the probability of GPCR heterodimer formation may be equal to or even higher than that of the corresponding homodimers and that specific properties of heterodimers can be revealed using a BRET-based ligand/receptor interaction assay.

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 β-arrestin (βarr) 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 βarr2-dependent signaling downstream of the truncated CXCR4 receptor. Indeed, the WHIM-associated receptor CXCR4¹⁰¹³ maintains association with βarr2 and triggers augmented and prolonged βarr2-dependent signaling, as revealed by ERK1/2 phosphorylation kinetics. Evidence is also provided that CXCR4¹⁰¹³-mediated chemotaxis critically requires βarr2, and disrupting the SHSK motif in the third intracellular loop of CXCR4¹⁰¹³ abrogates βarr2-mediated signaling, but not coupling to G proteins, and normalizes chemotaxis. We also demonstrate that CXCR4¹⁰¹³ spontaneously forms heterodimers with wild-type CXCR4. Accordingly, we propose a model where enhanced functional interactions between βarr2 and receptor dimers account for the altered responsiveness of WHIM leukocytes to CXCL12.

Leukocytes from individuals with warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a rare immunodeficiency, and bearing a wild-type CXCR4 ORF (WHIM^WT) display impaired CXCR4 internalization and desensitization upon exposure to CXCL12. The resulting enhanced CXCR4-dependent responses, including chemotaxis, probably impair leukocyte trafficking and account for the immunohematologic clinical manifestations of WHIM syndrome. We provided here evidence that GPCR kinase-3 (GRK3) specifically regulates CXCL12-promoted internalization and desensitization of CXCR4. GRK3-silenced control cells displayed altered CXCR4 attenuation and enhanced chemotaxis, as did WHIM^WT cells. These findings identified GRK3 as a negative regulator of CXCL12-induced chemotaxis and as a candidate responsible for CXCR4 dysfunction in WHIM^WT leukocytes. Consistent with this, we showed that GRK3 overexpression in both leukocytes and skin fibroblasts from 2 unrelated WHIM^WT patients restored CXCL12-induced internalization and desensitization of CXCR4 and normalized chemotaxis. Moreover, we found in cells derived from one patient a profound and selective decrease in GRK3 products that probably resulted from defective mRNA synthesis. Taken together, these results have revealed a pivotal role for GRK3 in regulating CXCR4 attenuation and have provided a mechanistic link between the GRK3 pathway and the CXCR4-related WHIM^WT disorder.