Ligand-independent CXCR2 Dimerization

Abstract

and the ʈIstituto di Neurobiologia Consiglio Nazionale delle Ricerche, Rome 00137, Italy Homo-and hetero-oligomerization have been reported for several G protein-coupled receptors (GPCRs). The CXCR2 is a GPCR that is activated, among the others, by the chemokines CXCL8 (interleukin-8) and CXCL2 (growth-related gene product ␤) to induce cell chemotaxis. We have investigated the oligomerization of CXCR2 receptors expressed in human embryonic kidney cells and generated a series of truncated mutants to determine whether they could negatively regulate the wild-type (wt) receptor functions. CXCR2 receptor oligomerization was also studied by coimmunoprecipitation of green fluorescent protein-and V5-tagged CXCR2. Truncated CXCR2 receptors retained their ability to form oligomers only if the region between the amino acids Ala-106 and Lys-163 was present. In contrast, all of the deletion mutants analyzed were able to form heterodimers with the wt CXCR2 receptor, albeit with different efficiency, competing for wt/wt dimer formation. The truncated CXCR2 mutants were not functional and, when coexpressed with wt CXCR2, interfered with receptor functions, impairing cell signaling and chemotaxis. When CXCR2 was expressed with the AMPA-type glutamate receptor GluR1, CXCR2 dimerization was again impaired in a dose-dependent way, and receptor functions were prejudiced. In contrast, CXCR1, a chemokine receptor that shares many similarities with CXCR2, did not dimerize alone or with CXCR2 and when coexpressed with CXCR2 did not impair receptor signaling and chemotaxis. The formation of CXCR2 dimers was also confirmed in cerebellar neuron cells. Taken together, we conclude from these studies that CXCR2 functions as a dimer and that truncated receptors negatively modulate receptor activities competing for the formation of wt/wt dimers.Homo-or heterodimerization of G protein-coupled receptors (GPCRs) 1 has recently emerged as a constitutive or ligandinduced property of several receptor types. Receptor oligomerization has functional implications in terms of cell surface expression, ligand binding, signaling, and receptor trafficking (1). Many different GPCRs have been proved to undergo homoor heterodimer formation; these include the receptors for dopamine which can form both homo-(2-4) and heterodimers with somatostatin receptors (5), angiotensin A1 and A2 (6), adrenergic (7,8), GABA B (9 -11), ␦-and -opioid (12-15), rhodopsin (16), mGlu (17), vasopressin V2 (18), vasopressin/ossitocin (19), muscarinic (20), glucagon (21), Ca 2ϩ (22) sphingosine 1-phosphate (23), and the chemokine CXCR4, CCR2, and CCR5 receptors (24 -28). The molecular mechanism of receptor oligomerization varies among different receptors and involves transmembrane (TM) domains as well as extramembrane regions. Cysteine residues in the fourth TM segment are responsible for D2 dopamine receptor dimerization (4), and disulfide bonds are also involved in the oligomerization of Ca 2ϩ (22), -opioid (13), sphingosine 1-phosphate (23), and V2 vasopressin receptors (18); sequen...

B1Bradykinin Receptor Homo-Oligomers in Receptor Cell Surface Expression and Signaling: Effects of Receptor Fragments

“…Truncation of the B 1 receptor revealed that homo-oligomerization of this receptor, as determined by coimmunoprecipitation of differentially tagged receptors, is dependent on an epitope(s) lo- cated between residue Leu 26 on top of transmembrane helix 1 and Val 71 at the C-terminal end of intracellular loop 1. This epitope may be similar to that in the yeast ␣-factor receptor (Overton et al, 2003), C5a receptor (Klco et al, 2003), and CCR2 receptor (Mellado et al, 2001), which apparently require helix 1 for homo-oligomerization, or the CXCR2 receptor, which requires helix 3 (Trettel et al, 2003). On the other hand, the dopamine D2 receptor forms a symmetric dimer interface involving helix 4 (Guo et al, 2003), and the B 2 bradykinin receptor subtype apparently requires an epitope in the N-terminal domain (AbdAlla et al, 1999).…”

Dynamic Regulation of CXCR1 and CXCR2 Homo- and Heterodimers

“…The balance between homo-and heterodimers might therefore have a role in CXCL8 function; indeed, via CXCR1, CXCL8 selectively activates phospholipase D and the neutrophil respiratory burst (14,24), whereas CXCR2 is involved in CXCL8-mediated neutrophil migration (50). Cumulative evidence indicates that the dimerization of chemokine receptors should no longer be a matter of debate (4,5,7,10,12,51,52). Efforts should focus on studying the dynamics and functional relevance of receptor homo-and heterodimerization; understanding these concepts will change our view of chemokine receptor structure and activation, which is likely to have substantial influence on drug development and screening.…”

“…These receptors have 77% overall sequence identity, but differ in their N-and C-terminal domains. Although initial reports suggested that only CXCR2 forms dimeric complexes (10,11), there is evidence that CXCR1 and CXCR2 form constitutive homo-and heterodimers with equal efficiency early in synthesis and maturation (12).…”

Identification of transmembrane domains that regulate spatial arrangements and activity of prokineticin receptor 2 dimers

“…The utility of deletion mutants has been demonstrated by different GPCR studies as receptor fragments can be expressed in a relatively native form (Marsango et al, 2011;Overton and Blumer, 2002;Trettel et al, 2003). Deletion mutants were: TM1-6 with a deletion in the region comprising the seventh TM helix, helix 8 and the C-terminal sequence, TM1-5 lacking the sixth and seventh TM helices, helix 8 and the C-terminal tail and TM1-4 missing the fifth, sixth and seventh TM helices, helix 8 and the C-terminal region.…”