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: | Pediani, Dr John and Lopez-Gimenez, Dr Juan and Canals Buj, Dr Meritxell and Milligan, Professor Graeme |
| Authors: | Lopez-Gimenez, J. F., Canals Buj, M., Pediani, J. D., and Milligan, G. |
| College/School: | College of Medical Veterinary and Life Sciences > School of Molecular Biosciences |
| 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 |
| Related URLs: |
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