Interactions of the alpha2A-adrenoceptor with multiple Gi-family G-proteins: studies with pertussis toxin-resistant G-protein mutants.
The alpha2A-adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of 'Gi-like' pertussis toxin-sensitive G-proteins. A number of members of this subfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual Gi-family G-proteins the porcine alpha2A-adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type Gi1alpha, Gi2alpha and Gi3alpha or mutations of each of these G-proteins in which the cysteine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue. The alpha2-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5'-[gamma-35thio]-triphosphate (GTP[35S]), when expressed without any additional G-protein. These effects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type Gi-like G-protein alpha-subunits resulted in enhanced agonist activation of the cellular G-protein population which was fully prevented by pretreatment with pertussis toxin. Co-expression of the receptor along with the cysteine-to-glycine mutations of Gi1alpha, Gi2alpha and Gi3alpha resulted in agonist stimulation of these G-proteins, which was as great as that of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed Gi-like G-proteins was resistant to pertussis toxin treatment. The Cys --> Gly mutations of Gi1alpha, Gi2alpha and Gi3alpha were each also able to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resistant mutant of Gi1alpha was correlated highly both with the level of expression of this G-protein and with the level of expression of the alpha2A-adrenoceptor. Half-maximal stimulation of high-affinity GTPase activity of the Cys --> Gly mutants of Gi1alpha, Gi2alpha and Gi3alpha required 10-15-fold higher concentrations of agonist than did stimulation of their wild-type counterparts, consistent with a model in which the affinity of functional interactions of the alpha2A-adrenoceptor with the wild-type G-protein is greater than with the pertussis toxin-resistant mutant G-protein.
Wise
A
A
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, U.K.
Watson-Koken
M A
MA
Rees
S
S
Lee
M
M
Milligan
G
G
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D000068438:Brimonidine Tartrate; D019556:COS Cells; D003001:Cloning, Molecular; D020558:GTP Phosphohydrolases / Q000032:analysis / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D037342:Pertussis Toxin; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D014162:Transfection / Q000235:genetics; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D037361:Adenylate Cyclase Toxin; D000316:Adrenergic alpha-Agonists; D000906:Antibodies; D010446:Peptide Fragments; D011810:Quinoxalines; D011942:Receptors, Adrenergic, alpha; D010566:Virulence Factors, Bordetella; D000068438:Brimonidine Tartrate; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases