The 1b-adrenoceptor exists as a higher-order oligomer: effective oligomerization is required for receptor maturation, surface delivery, and function

Lopez-Gimenez, J. F., Canals Buj, M., Pediani, J. D. and Milligan, G. (2007) The 1b-adrenoceptor exists as a higher-order oligomer: effective oligomerization is required for receptor maturation, surface delivery, and function. Molecular Pharmacology, 71(4), pp. 1015-1029. (doi:10.1124/mol.106.033035)

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Publisher's URL: http://dx.doi.org/10.1124/mol.106.033035

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 α1b-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 N-glycosylation 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 α1b-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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Milligan, Professor Graeme and Canals Buj, Dr Meritxell and Pediani, Dr John and Lopez-Gimenez, Dr Juan
Authors: Lopez-Gimenez, J. F., Canals Buj, M., Pediani, J. D., and Milligan, G.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Molecular Pharmacology
Journal Abbr.:Mol. Pharmacol.
Publisher:American Society for Pharmacology and Experimental Therapeutics
ISSN:0026-895X
ISSN (Online):1521-0111
Published Online:12 January 2007
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
389781The quaternary structure of G-protein coupled receptors - implications for function drug design. Programme grant support renewalGraeme MilliganMedical Research Council (MRC)G9811527Institute of Neuroscience and Psychology
242491Quantitative Analysis of the Effects of Receptor and G-Protein Mutations and Polymorphisms on Signal InitiationGraeme MilliganMedical Research Council (MRC)G9811527Institute of Neuroscience and Psychology
242492Quantitative Analysis of the Effects of Receptor and G-Protein Mutations and Polymorphisms on Signal InitiationGraeme MilliganMedical Research Council (MRC)G9811527Institute of Neuroscience and Psychology