The effect of site-directed mutagenesis of two transmembrane serine residues on agonist-specific coupling of a cloned human alpha2A-adrenoceptor to adenylyl cyclase.
1. The effects of substitution of the Ser200 and Ser204 residues with alanine on the signalling properties of the cloned human alpha2A-adrenoceptor, stably expressed in Chinese hamster ovary (CHO) cell lines, have been investigated using noradrenaline and the structural isomers of octopamine. 2. The Ser-->Ala200 or the Ser-->Ala204 mutant forms of the alpha2A-adrenoceptor, when expressed in cells in the absence of pertussis toxin pretreatment, are two orders of magnitude more sensitive to inhibition of cyclic AMP production by (+/-)-para-octopamine and (+/-)-meta-octopamine, respectively, than cells expressing the wild-type receptor. Binding studies indicate that the effects are not due to an increased agonist affinity for the mutant receptors and that they are likely to be due to agonist-mediated conformational changes in receptor structure. 3. After incubation with pertussis toxin, (+/-)-meta-octopamine (100 microM and above) produced a stimulation of cyclic AMP levels in cells expressing the Ser-->Ala204 mutant form of the alpha2A-adrenoceptor but showed no stimulation in cells expressing the Ser-->Ala200 mutant receptor. Under these conditions (+/-)-para-octopamine did not produce any increases in cyclic AMP production in cells expressing either of the mutant receptor forms or the wild-type receptor. 4. The results emphasise the importance of the Ser200 and Ser204 residues of the alpha2A-adrenoceptor in exerting an inhibitory influence on the ability of (+/-)-para-octopamine and (+/-)-meta-octopamine respectively, to induce a receptor-agonist conformation capable of inhibiting forskolin-stimulation of cyclic AMP levels. 5. It is clear that Ser204 also prevents meta-octopamine from generating a receptor-agonist conformation that can increase cyclic AMP levels, emphasising the importance of this residue in the agonist-specific coupling of this receptor to different second messenger systems.
Rudling
J E
JE
The Babraham Institute Laboratory of Molecular Signaling, Department of Zoology, University of Cambridge.
Kennedy
K
K
Evans
P D
PD
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009638:Norepinephrine / Q000494:pharmacology; D009655:Octopamine / Q000031:analogs & derivatives / Q000494:pharmacology; D037342:Pertussis Toxin; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry / Q000502:physiology*; D012694:Serine; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000316:Adrenergic alpha-Agonists; D018341:Receptors, Adrenergic, alpha-2; D010566:Virulence Factors, Bordetella; D009655:Octopamine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012694:Serine; C005310:norfenefrine; D000242:Cyclic AMP; D037342:Pertussis Toxin; D000262:Adenylyl Cyclases; D009638:Norepinephrine