Received for publication November 29, 2006.
Revised January 11, 2007.
Accepted for publication January 12, 2007.

The _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 signalling function. Although these mutations prevented cell surface delivery, bi-molecular fluorescence complementation studies indicated they did not ablate protein-protein interactions and confirmed endoplasmic reticulum/Golgi retention of the transmembrane domain I + transmembrane domain IV mutated receptor. The transmembrane domain I + 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 whilst restricting mutation to transmembrane domain IV did result in reduced 3-FRET. Mutations in either transmembrane domain I or transmembrane domain IV were, however, sufficient to eliminate cell surface delivery. Terminal N-glycosylated is insufficient to determine cell surface delivery because both the 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.

Key words: Adrenergic, Gq/11 family, Receptor synthesis/trafficking, Sequestration/Internalization, Fluorescence techniques

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