Multiple interactions between transmembrane helices generate the oligomeric alpha1b-adrenoceptor.
Combinations of coimmunoprecipitation, single-cell fluorescence resonance energy transfer, and cell-surface time-resolved fluorescence resonance energy transfer demonstrated protein-protein interactions and quaternary structure for the alpha(1b)-adrenoceptor. Self-association of transmembrane domain 1 and its interaction with the full-length receptor indicated a symmetrical interface provided by this domain. Lack of effect of mutation of the glycophorin-A dimerization-like region within this helix demonstrated that this did not provide the molecular mechanism. Multiple interactions were observed between the alpha(1b)-adrenoceptor and fragments derived from its sequence. Fragments comprising transmembrane domains 3 and 4 and transmembrane domains 5 and 6, but not transmembrane domain 7, were also able to interact with the full-length receptor. Transmembrane domain 7 failed to interact significantly with any element of the receptor and was not transported to the cell surface after coexpression with the full-length receptor. Symmetrical interactions were also noted between fragments incorporating transmembrane domain 4, but this segment of the receptor failed to interact with transmembrane domains 1 and 2 or transmembrane domains 5 and 6. Time-resolved fluorescence resonance energy transfer studies were also consistent with contributions of transmembrane domains 1 and/or 2 and transmembrane domains 3 and/or 4 to protein-protein interactions within the quaternary structure of the alpha(1b)-adrenoceptor, and with a contribution of transmembrane domains 5 and/or 6. These data are consistent with a complex oligomeric quaternary structure of the alpha(1b)-adrenoceptor in which major, symmetrical interactions may define intradimeric contacts with other contributions, providing interdimer contacts to generate oligomeric complexes akin to those observed for murine rhodopsin. A model derived from this was developed.
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
Juan J
JJ
Carrillo
Juan F
JF
López-Giménez
Graeme
G
Milligan
D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D019281:Dimerization; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry / Q000378:metabolism*
C547716:ADRA1B protein, human; D008565:Membrane Proteins; D018340:Receptors, Adrenergic, alpha-1