The α_1b-Adrenoceptor Exists as a Higher-Order Oligomer: Effective Oligomerization Is Required for Receptor Maturation, Surface Delivery, and Function

Abstract

Approaches to identify G protein-coupled receptor oligomers rather than dimers have been lacking. Using concatamers of fluorescent proteins, we established conditions to monitor sequential three-color fluorescence resonance energy transfer (3-FRET) and used these to detect oligomeric complexes of the ␣ 1b -adrenoceptor in single living cells. Mutation of putative key hydrophobic residues in transmembrane domains I and IV resulted in substantial reduction of sequential 3-FRET and was associated with lack of protein maturation, prevention of plasma membrane delivery, and elimination of signaling function. Although these mutations prevented cell surface delivery, bimolecular fluorescence complementation studies indicated that they did not ablate protein-protein interactions and confirmed endoplasmic reticulum/Golgi retention of the transmembrane domain I plus transmembrane domain IV mutated receptor. The transmembrane domain I plus transmembrane domain IV mutated receptor was a "dominant-negative" in blocking cell surface delivery of the wild-type receptor. Mutations only in transmembrane domain I did not result in a reduction in 3-FRET, whereas restricting mutation to transmembrane domain IV did result in reduced 3-FRET. Mutations in either transmembrane domain I or transmembrane domain IV, however, were sufficient to eliminate cell surface delivery. Terminal Nglycosylation is insufficient to determine cell surface delivery because both transmembrane domain I and transmembrane domain IV mutants matured as effectively as the wild-type receptor. These data indicate that the ␣ 1b -adrenoceptor is able to form oligomeric rather than only simple dimeric complexes and that disruption of effective oligomerization by introducing mutations into transmembrane domain IV has profound consequences for cell surface delivery and function.

Activation of a Dimeric Metabotropic Glutamate Receptor by Intersubunit Rearrangement

“…If this is also the case for the full-length mGluR at the cell surface, this could lead to an alternative and maybe more complex activation mechanism. Indeed, higher order oligomers have been observed recently for rhodopsin (41) and ␣-adrenergic receptors (42). Here, using fluorophore-labeled antibodies, we have shown that no significant FRET could be measured between mGluR dimers, whereas a high FRET signal was measured between the subunits within a dimer.…”

“…mGlu5 Is a Dimer, Not a Higher Order Oligomer-At least some GPCRs may form not only dimers, but also higher order oligomers (41,42). If this occurs also for mGluRs, this would substantially complicate our analysis of their intramolecular signal transduction.…”

Identification of a serotonin/glutamate receptor complex implicated in psychosis

“…Mouse lines, treatment protocols, behavioural studies, dissections, and primary neuronal cultures, approved by Institutional Use and Care Committees, have been previously described 8,9 . Protocols used for FISH 8 , binding assays 8 , real-time PCR 8 , FRET 17 and co-immunoprecipitation 17 were performed as previously described or with minor modifications. Epitope tagged, BRET 2 , FRET and chimera receptor constructs were generated using standard cloning techniques and were confirmed by sequencing.…”

“…Recent studies have demonstrated that some GPCRs belonging to the same sequence classes can form dimers 16 or, potentially, higher-order oligomers 17 . Although the 2AR and mGluR2 belong to different GPCR classes, we established the existence of 2AR/mGluR2 heterocomplexes by several methods: co-immunoprecipitation of human brain cortex samples (Fig.…”

G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo

“…Although the GnRH receptor was one of the very first GPCRs shown to oligomerize at the PM as part of normal receptor function (Conn et al, 1982;Cornea et al, 2001), oligomerization in the ER-Golgi complex at the time of nascent protein synthesis and routing to the cell surface is a relatively new concept for GPCRs and suggests that there may also be a function for oligomerization at that early stage (Lee et al, 2000;Lopez-Gimenez et al, 2007;Pin et al, 2007). Perhaps oligomerization of two or more receptors acts to hide exposed hydrophobic surfaces or ER retention motifs that would otherwise signal an improperly folded receptor and be recognized as such by the quality control apparatus of the cell.…”