No abstract

Although homodimerization has been demonstrated for a large number of G protein-coupled receptors (GPCRs), no general role has been attributed to this process. Because it is known that oligomerization plays a key role in the quality control and endoplasmic reticulum (ER) export of many proteins, we sought to determine if homodimerization could play such a role in GPCR biogenesis. Using the ␤2-adrenergic receptor (␤2AR) as a model, cell fractionation studies revealed that receptor homodimerization is an event occurring as early as the ER. Supporting the hypothesis that receptor homodimerization is involved in ER processing, ␤2AR mutants lacking an ER-export motif or harboring a heterologous ER-retention signal dimerized with the wild-type receptor and inhibited its trafficking to the cell surface. Finally, in addition to inhibiting receptor dimerization, disruption of the putative dimerization motif, 276 GXXXGXXXL 284, prevented normal trafficking of the receptor to the plasma membrane. Taken together, these data indicate that ␤2AR homodimerization plays an important role in ER export and cell surface targeting.Recent studies have established that G protein-coupled receptors (GPCRs), 1 much like other membrane receptors, form both homodimers and heterodimers (1, 2). Various functions have been attributed to the dimerization between different receptor subtypes. For instance, heterodimerization has been proposed to promote the formation of receptors with unique pharmacological properties, contributing to the pharmacological diversity of GPCRs (3-5). Changes in G protein coupling specificity (6) as well as altered receptor endocytosis (3, 4, 6 -9) have also been proposed to result from receptor heterodimerization. For the metabotropic GABAb receptor, heterodimerization between GABAb-R1 and GABAb-R2 is an obligatory step for cell surface expression of a functional receptor (10,11). This was shown to result from the masking of an endoplasmic reticulum (ER) retention signal found in the C terminus of GABAb-R1, presumably through the formation of a coiled-coil domain between the C termini of the two receptor subtypes (12). The obligatory nature of this heterodimerization for cell surface trafficking and signaling was considered for a long time as a particularity of the GABAb receptor, which belongs to a GPCR subfamily (family 3) containing only a few members (13). However, a recent study indicated that the heterodimerization between two closely related members of family 1 GPCRs, the adrenergic ␣ 1D and ␣ 1B receptors, may be a prerequisite for the proper cell surface expression of the ␣ 1D subtype in both experimental models and real tissues (14).So far, despite the numerous examples of receptor homodimerization reported in the literature (15, 16), the biological function of this phenomenon has remained elusive, probably because it is extremely difficult to experimentally differentiate the dimer properties of identical receptor subtypes from those of monomers. However, a growing number of observations indicates that GPC...

The ability of the closely related ␤ 2 -and ␤ 3 -adrenergic receptors (AR) to form hetero-oligomers was assessed by bioluminescence resonance energy transfer. Quantitative bioluminescence resonance energy transfer titration curves revealed that the ␤ 2 AR has identical propensity to hetero-oligomerize with the ␤ 3 AR than to form homo-oligomers. To determine the influence of heterooligomerization, a HEK293 cell line stably expressing an excess of ␤ 3 AR over ␤ 2 AR was generated so that all ␤ 2 AR are engaged in hetero-oligomerization with ␤ 3 AR, providing a tool to study the effect of hetero-oligomerization on ␤ 2 AR function in the absence of any ␤ 2 AR homooligomer. The hetero-oligomerization had no effect on the ligand binding properties of various ␤ 2 AR ligands and did not affect the potency of isoproterenol to stimulate adenylyl cyclase. Despite the unaltered ligand binding properties of the ␤ 2/3 AR hetero-oligomer, the stable association of the ␤ 2 AR with the ␤ 3 AR completely blocked agonist-stimulated internalization of the ␤ 2 AR. Given that the ␤ 3 AR is resistant to agonist-promoted endocytosis, the results indicate that the ␤ 3 AR acted as a dominant negative of the ␤ 2 AR endocytosis process. Consistent with this notion, the ␤ 2/3 AR hetero-oligomer displayed a lower propensity to recruit ␤-arrestin-2 than the ␤ 2 AR. The hetero-oligomerization also led to a change in G protein coupling selectivity. Indeed, in contrast to ␤ 2 AR and ␤ 3 AR, which regulate adenylyl cyclase and extracellular signal-regulated kinase activity through a coupling to G s and G i/o , no G i/o coupling was observed for the ␤ 2/3 AR hetero-oligomer. Together, these results demonstrate that hetero-oligomerization between ␤ 2 AR and ␤ 3 AR forms a ␤-adrenergic signaling unit that possesses unique functional properties.

In most cases, nephrogenic diabetes insipidus results from mutations in the V2 vasopressin receptor (V2R) gene that cause intracellular retention of improperly folded receptors. We previously reported that cell permeable V2R antagonists act as pharmacological chaperones that rescue folding, trafficking, and function of several V2R mutants. More recently, the vasopressin antagonist, SR49059, was found to be therapeutically active in nephrogenic diabetes insipidus patients. Three of the patients with positive responses harbored the mutation R137H, previously reported to lead to constitutive endocytosis. This raises the possibility that, instead of acting as a pharmacological chaperone by favoring proper maturation of the receptors, SR49059 could mediate its action on R137H V2R by preventing its endocytosis. Here we report that the beta-arrestin-mediated constitutive endocytosis of R137H V2R is not affected by SR49059, indicating that the functional rescue observed does not result from a stabilization of the receptor at the cell surface. Moreover, metabolic labeling revealed that R137H V2R is also poorly processed to the mature form. SR49059 treatment significantly improved its maturation and cell surface targeting, indicating that the functional rescue of R137H V2Rs results from the pharmacological chaperone action of the antagonist.

Palmitoylation is unique among lipid modifications in that it is reversible. In recent years, dynamic palmitoylation of G protein ␣ subunits and of their cognate receptors has attracted considerable attention. However, very little is known concerning the acylation/deacylation cycle of the proteins in relation to their activity status. In particular, the relative contribution of the activation and desensitization of the signaling unit to the regulation of the receptors and G proteins palmitoylation state is unknown. To address this issue, we took advantage of the fact that a fusion protein composed of the stimulatory ␣ subunit of trimeric G protein (G␣ s ) covalently attached to the ␤ 2 -adrenergic receptor (␤ 2 AR) as a carboxyl-terminal extension (␤ 2 AR-G␣ s ) can be stimulated by agonists but does not undergo rapid inactivation, desensitization, or internalization. When expressed in Sf9 cells, both the receptor and the G␣ s moieties of the fusion protein were found to be palmitoylated via thioester linkage. Stimulation with the ␤-adrenergic agonist isoproterenol led to a rapid depalmitoylation of both the ␤ 2 AR and G␣ s and inhibited repalmitoylation. The extent of depalmitoylation induced by a series of agonists was correlated (0.99) with their intrinsic efficacy to stimulate the adenylyl cyclase activity. However, forskolin-stimulated cAMP production did not affect the palmitoylation state of ␤ 2 AR-G␣ s , indicating that the agonist-promoted depalmitoylation is linked to conformational changes and not to second messenger generation. Given that, upon activation, the fusion protein mimics the activated receptor-G protein complex but cannot undergo desensitization, the data demonstrate that early steps in the activation process lead to the depalmitoylation of both receptor and G protein and that repalmitoylation requires later events that cannot be accommodated by the activated fusion protein.Palmitoylation is a post-translational modification that is limited to a small subset of cellular proteins among which proteins involved in signal transduction are prevalent (1). This thioesterification of cysteine residues by palmitate distinguishes itself from other lipid modifications such as prenylation and myristoylation by its reversibility. Indeed, in contrast to myristoyl and prenyl moieties that are added co-translationally and generally remain attached to the proteins until the protein gets degraded, the protein-bound palmitate is added post-translationally and turns over more rapidly than the protein itself (2-4). Moreover, the palmitoylation state of several proteins has been shown to be dynamically regulated. In particular, biological regulation of the palmitoylation state of heterotrimeric G proteins and of their cognate receptors has been demonstrated (5-12).Activation of G␣ s 1 through receptor stimulation, following direct activation with aluminum fluoride and cholera toxin or as a result of site-directed mutagenesis, has been shown to lead to an increased incorporation of [ 3 H]palmitate into G␣ s duri...