Properties and regulation of the coupling to adenylate cyclase of secretin receptors stably transfected in Chinese hamster ovary cells.
The binding properties, coupling to adenylate cyclase, and desensitization of secretin receptors stably expressed in transfected Chinese hamster ovary (CHO) cells were compared in two clones expressing high (CHO-SnR-c5, 450 +/- 80 fmol/mg of protein) and low (CHO-SnR-c1, 40 +/- 25 fmol/mg of protein) receptor densities. The Kd values for receptor occupancy by secretin, selected analogues, and fragments were identical in CHO-SnR-c1 and -c5 cells and identical to those described for native receptors from NG 108-15 cells. The Kact values for adenylate cyclase stimulation were identical to the Kd values in CHO-SnR-c1 cells but 5-10-fold higher than those in CHO-SnR-c5 cells. The Kact values in both CHO-SnR-c1 and -c5 cell lines were reduced in the presence of the nonhydrolyzable GTP derivative guanosine-5'-(beta, gamma-imido)triphosphate and after pretreatment of the cells with cholera toxin. Preincubation of both CHO-SnR-c1 and -c5 cell lines with secretin for 24 hr reduced their binding capacity and reduced secretin efficacy in CHO-SnR-c1 cells and secretin potency in CHO-SnR-c5 cells. These results suggest efficient coupling of the secretin receptor to the adenylate cyclase machinery and the existence of spare receptors in the clone expressing higher receptor density. Pretreatment of the two cell lines with the reducing agent dithiothreitol reduced the binding capacity and induced the appearance of a low affinity binding component. In both cell lines, dithiothreitol pretreatment decreased secretin potency but not secretin efficacy, suggesting the necessity of integrity of the disulfide bridges for optimal receptor recognition.
Vilardaga
J P
JP
Laboratory of Biochemistry and Nutrition, Faculty of Medicine, Université Libre de Bruxelles, Belgium.
Ciccarelli
E
E
Dubeaux
C
C
De Neef
P
P
Bollen
A
A
Robberecht
P
P
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004229:Dithiothreitol / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012633:Secretin / Q000378:metabolism* / Q000502:physiology; D014162:Transfection
D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; D011994:Recombinant Proteins; C029937:secretin receptor; D012633:Secretin; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D002772:Cholera Toxin; D000262:Adenylyl Cyclases; D004229:Dithiothreitol