Dimerization of the δ Opioid Receptor:

Members of the G-protein-coupled receptor (GPCR)family are involved in most aspects of higher eukaryote biology, and mutations in their coding sequence have been linked to several diseases. In the present study, we report that mutant GPCR can affect the functional properties of the co-expressed wild type (WT) receptor. Mutants of the human platelet-activating factor receptor that fail to show any detectable ligand binding (N285I and K298stop) or coupling to a G-protein (D63N, D289A, and Y293A) were co-expressed with the WT receptor in Chinese hamster ovary and COS-7 cells. In this context, N285I and K298stop mutant receptors inhibited 3 H-WEB2086 binding and surface expression. Co-transfection with D63N resulted in a constitutively active receptor phenotype. Platelet-activating factor-induced inositol phosphate production in cells transfected with a 1:1 ratio of WT:D63N was higher than with the WT cDNA alone but was abolished with a 1:3 ratio. We confirmed that these findings could be extended to other GPCRs by showing that co-expression of the WT C-C chemokine receptor 2b with a carboxyl-terminal deletion mutant (K311stop), resulted in a decreased affinity and responsiveness to MCP-1. A better understanding of this phenomenon could lead to important tools for the prevention or treatment of certain diseases.Certain ligands can assume distinct functions in different tissues. For example, platelet-activating factor (PAF) 1 is involved in embryogenesis as well as in modulation of a variety of functions of the immune and central nervous systems (1-3). With respect to PAF, for which the only known receptor is a member of the G-protein-coupled receptor (GPCR) family, the diversity of responses generated is likely a result of the receptor coupling to different signaling pathways in the target cells (3, 4). On the other hand, for other ligands such as adrenaline and dopamine, the cell can also determine the specificity of its response by the differential expression of receptor subtypes with distinct characteristics of binding, coupling, and desensitization (5). Subtypes of a given receptor can be derived from distinct genes or generated by alternative splicing. Divergence between receptor isoforms, as for the prostaglandin (EP) and the MCP-1 (CCR2) receptors, is most often limited to the carboxyl-terminal cytoplasmic tail, a region that is potentially involved in G-protein coupling, internalization, and down-regulation of the receptors (6, 7).Alternative splicing can also lead to the formation of nonfunctional receptors or receptors with certain functions that are greatly modified (8, 9). Individually, these receptors are not involved in signaling, but some of them can show dominantnegative properties when co-expressed with a functional subtype. It has recently been demonstrated that the expression of a truncated isoform of the human gonadotropin-releasing hormone receptor could affect the extent of agonist-specific cellular response by inhibiting the cell surface expression of the functional isoform (10). A similar e...

mentioning

confidence: 99%

Selective Modulation of Wild Type Receptor Functions by Mutants of G-Protein-coupled Receptors

Gouill

Parent

Caron

et al. 1999

“…These findings suggest that significant functional interactions may take place between -and ␦-opioid receptors in specific neuronal pathways in brain. We and many other groups have described dimers and oligomers for many GPCRs 1 mediating the actions of neurotransmitters, such as dopamine (9 -12) and serotonin (13), and for neuropeptide receptors, such as vasopressin (14) and opioid peptides (15). Dimerization of the ␦-opioid receptor has been suggested to have an important role in activation and internalization of the receptor (15).…”

mentioning

confidence: 99%

Oligomerization of μ- and δ-Opioid Receptors

George

Fan

Xie³

et al. 2000

510

154

The existence of dimers and oligomers for many G protein-coupled receptors has been described by us and others. Since many G protein-coupled receptor subtypes are highly homologous to each other, we examined whether closely related receptors may interact with each other directly and thus have the potential to create novel signaling units. Using -and ␦-opioid receptors, we show that each receptor expressed individually was pharmacologically distinct and could be visualized following electrophoresis as monomers, homodimers, homotetramers, and higher molecular mass oligomers. When -and ␦-opioid receptors were coexpressed, the highly selective synthetic agonists for each had reduced potency and altered rank order, whereas endomorphin-1 and Leu-enkephalin had enhanced affinity, suggesting the formation of a novel binding pocket. No heterodimers were visualized in the membranes coexpressing -and ␦-receptors by the methods available. However, heterooligomers were identified by the ability to co-immunoprecipitate -receptors with ␦-receptors and vice versa using differentially epitope-tagged receptors. In contrast to the individually expressed -and ␦-receptors, the coexpressed receptors showed insensitivity to pertussis toxin and continued signal transduction, likely due to interaction with a different subtype of G protein.In this study, we provide, for the first time, evidence for the direct interaction of -and ␦-opioid receptors to form oligomers, with the generation of novel pharmacology and G protein coupling properties.Opioid receptors have distinct pharmacological profiles and discrete but overlapping distributions in brain. The relatively recent cloning of opioid receptors has established that the products of three genes form the known subtypes (the -, ␦-, and -opioid receptors) that interact with the complex family of endogenous opioid peptides (reviewed in Ref. 1). The endogenous opioid peptide-receptor systems mediate important physiological functions related to pain perception, locomotion, motivation, reward, autonomic function, immunomodulation, and hormone secretion.The analysis of the contribution of each receptor type to the various opioid functions documented has been limited by the selectivity and cross-reactivity of the available opioid ligands and the postulation that multiple receptor subtypes are present. Since the cloning of the opioid receptors, the individual pharmacological and biochemical profiles of the -, ␦-, and -opioid receptors have been better defined; however, there are many aspects of opioid receptor biology that still remain poorly understood. A major problem that still remains is that the pharmacology of opioid receptors in brain tissue predicts a greater number of receptor subsites than revealed by opioid receptor cloning (1, 2). One possible explanation may be that the precise receptor-effector interactions present in endogenous brain regions may have not been adequately replicated in the heterologous expression systems in which the cloned receptors have been studied or, alternativel...

“…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); sequences in the N-terminal extracellular domains are involved in mGluR (17) and CCR5 (24 -27) homodimer formation; the C-terminal region is essential for ␦-opioid receptor dimerization (12). The seventh TM domain controls noncovalent hydrophobic interactions for adrenergic receptors, which also require receptor glycosylation (7,8).…”

mentioning

confidence: 99%

“…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)(13)(14)(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.…”

mentioning

confidence: 99%

Ligand-independent CXCR2 Dimerization

Trettel

Bartolomeo

Lauro

et al. 2003

103

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...