Stable expression of normal and mutant human ACTH receptor: study of ACTH binding and coupling to adenylate cyclase.
Point mutations of the human ACTH receptor have been reported in some patients with a familial glucocorticoid deficiency syndrome. To demonstrate that these mutations were responsible for the disease, it was necessary to develop a model in which characteristics of normal and mutant receptors could be studied. We have developed a stable expression model in order to characterize the human ACTH receptor by binding studies and functional coupling to adenylate cyclase. After confirmation of the stable integration of receptor constructs, ACTH dose-responses for the production of cAMP were carried out. The EC50 for ACTH were 2.9 +/- 0.2 x 10(-10) M and 2.4 +/- 0.8 x 10(-10) M, respectively, for two different clones stably expressing the normal human ACTH receptor. EC50 calculated for clones expressing either one of the two studied mutant receptors (C251F and D107N) were increased: 4.1 +/- 0.9 x 10(-9) M and 6.4 +/- 1.3 x 10(-9) M respectively. These values were similar to that obtained with M3 parental cells (4.7 +/- 0.8 x 10(-9) M). Binding studies were performed on the same clones. Scatchard analysis showed that clones expressing the normal receptor possessed high affinity binding sites for ACTH, with K(d) = 5.8 +/- 2.4 x 10(-10) M and 6.9 +/- 3.6 x 10(-10) M, respectively, for the two different studied clones. A second type of sites, with low affinity (K(d) around 10(-8) M), was also present. There was no ACTH binding to the high affinity binding sites for the two clones expressing either one of the mutant receptors. An impaired binding of ACTH to its receptors is then responsible for the absence of biological response to ACTH in patients carrying these mutant ACTH receptors.
Naville
D
D
INSERM-INRA U418, Hôpital Debrousse, Lyon, France.
Barjhoux
L
L
Jaillard
C
C
Saez
J M
JM
Durand
P
P
Bégeot
M
M
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000324:Adrenocorticotropic Hormone / Q000378:metabolism*; D001483:Base Sequence; D002999:Clone Cells; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D007700:Kinetics; D017354:Point Mutation; D018043:Receptors, Corticotropin / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D018043:Receptors, Corticotropin; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP; D000262:Adenylyl Cyclases