TY - JOUR
T1 - Divergent structural requirements exist for calcitonin receptor binding specificity and adenylate cyclase activation
AU - Houssami, S.
AU - Findlay, D. M.
AU - Brady, C. L.
AU - Martin, T. J.
AU - Epand, R. M.
AU - Moore, E. E.
AU - Murayama, E.
AU - Tamura, T.
AU - Orlowski, R. C.
AU - Sexton, P. M.
PY - 1995/1/1
Y1 - 1995/1/1
N2 -
The basis of the high potency of salmon calcitonin (sCT) in radioligand binding competition and cAMP accumulation studies with cloned calcitonin (CT) receptors from rats, pigs, and humans was examined using two sets of CT analogues, i.e., chimeric sCT/human CT (hCT) analogues and analogues of sCT with differing capacities to form an amphipathic α-helix. In competition for
125
I-sCT binding the following relative specificities were observed for the chimeric peptides: rat C1a CT receptor, sCT ≥ (1-16)hCT/(17-32)sCT (ACT- 15) > (1-16)sCT/(17-32)hCT (ACT-27); rat C1b CT receptor, sCT >> ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 > ACT-27; porcine CT receptor, sCT > ACT- 27 > ACT-15. In contrast, in ligand-induced cAMP accumulation studies the relative efficacies were as follows: rat C1a CT receptor, sCT = ACT-15 > ACT- 27; rat C1b CT receptor, sCT = ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 ≥ ACT-27; porcine CT receptor, sCT = ACT-15 = ACT-27. The data demonstrate that residues present in the carboxyl-terminal half of sCT are more important for binding competition with the rat C1a, rat C1b, and human CT receptors, whereas residues in the amino-terminal half of sCT are more important for binding competition with the porcine CT receptor. Carboxyl-terminal sCT residues are also important for full potency in adenylate cyclase activation with the rat C1a and rat C1b CT receptors but are less important for activation via the hCT receptor. The disparity in the relative potencies of the peptides in studies of binding competition and cAMP accumulation is suggestive of significant differences in the relative affinities of the peptides for active and inactive conformations of the CT receptor. The use of sCT analogues with varying capacities to form α-helices also revealed divergence in the responses of different receptors. This was most apparent for the stimulation of CAMP production by the rat receptor isoforms C1a and C1b. In cells expressing the C1a receptor, the helical analogues sCT and des- Ser
2
-sCT were equipotent with [Gly
8
]-des-Leu
19
-sCT and des-1-amino- [Ala
1,7
,Gly
8
]-des-Leu
19
sCT, analogues that have reduced or absent helical structure, respectively. In contrast, the nonhelical analogues were 100-1000-fold less potent than sCT and des-Ser
2
-sCT at the C1b receptor. In general, reduction in the ability of sCT analogues to form helix structures had a greater impact on the potency of the analogues in competition for
125
I-sCT binding than in cAMP accumulation. The absence or reduction of helix-forming potential had much less impact on the interaction of the analogues with the cloned hCT receptor in both assay systems but also had minimal impact on the activation of the rat C1a receptor, as assessed by cAMP responses.
AB -
The basis of the high potency of salmon calcitonin (sCT) in radioligand binding competition and cAMP accumulation studies with cloned calcitonin (CT) receptors from rats, pigs, and humans was examined using two sets of CT analogues, i.e., chimeric sCT/human CT (hCT) analogues and analogues of sCT with differing capacities to form an amphipathic α-helix. In competition for
125
I-sCT binding the following relative specificities were observed for the chimeric peptides: rat C1a CT receptor, sCT ≥ (1-16)hCT/(17-32)sCT (ACT- 15) > (1-16)sCT/(17-32)hCT (ACT-27); rat C1b CT receptor, sCT >> ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 > ACT-27; porcine CT receptor, sCT > ACT- 27 > ACT-15. In contrast, in ligand-induced cAMP accumulation studies the relative efficacies were as follows: rat C1a CT receptor, sCT = ACT-15 > ACT- 27; rat C1b CT receptor, sCT = ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 ≥ ACT-27; porcine CT receptor, sCT = ACT-15 = ACT-27. The data demonstrate that residues present in the carboxyl-terminal half of sCT are more important for binding competition with the rat C1a, rat C1b, and human CT receptors, whereas residues in the amino-terminal half of sCT are more important for binding competition with the porcine CT receptor. Carboxyl-terminal sCT residues are also important for full potency in adenylate cyclase activation with the rat C1a and rat C1b CT receptors but are less important for activation via the hCT receptor. The disparity in the relative potencies of the peptides in studies of binding competition and cAMP accumulation is suggestive of significant differences in the relative affinities of the peptides for active and inactive conformations of the CT receptor. The use of sCT analogues with varying capacities to form α-helices also revealed divergence in the responses of different receptors. This was most apparent for the stimulation of CAMP production by the rat receptor isoforms C1a and C1b. In cells expressing the C1a receptor, the helical analogues sCT and des- Ser
2
-sCT were equipotent with [Gly
8
]-des-Leu
19
-sCT and des-1-amino- [Ala
1,7
,Gly
8
]-des-Leu
19
sCT, analogues that have reduced or absent helical structure, respectively. In contrast, the nonhelical analogues were 100-1000-fold less potent than sCT and des-Ser
2
-sCT at the C1b receptor. In general, reduction in the ability of sCT analogues to form helix structures had a greater impact on the potency of the analogues in competition for
125
I-sCT binding than in cAMP accumulation. The absence or reduction of helix-forming potential had much less impact on the interaction of the analogues with the cloned hCT receptor in both assay systems but also had minimal impact on the activation of the rat C1a receptor, as assessed by cAMP responses.
UR - http://www.scopus.com/inward/record.url?scp=0028931438&partnerID=8YFLogxK
M3 - Article
C2 - 7723741
AN - SCOPUS:0028931438
SN - 0026-895X
VL - 47
SP - 798
EP - 809
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 4
ER -