Circular dichroic evidence for an ordered sequence of ligand/binding site interactions in the catalytic reaction of the cAMP-dependent protein kinase

Biochemistry ◽  
1985 ◽  
Vol 24 (12) ◽  
pp. 2967-2973 ◽  
Author(s):  
Jennifer Reed ◽  
Volker Kinzel ◽  
Bruce E. Kemp ◽  
Hueng Chin Cheng ◽  
Donal A. Walsh
Keyword(s):  
Protein Kinase ◽  
Ligand Binding ◽  
Binding Site ◽  
Catalytic Reaction ◽  
Ligand Binding Site ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Circular Dichroic

scholarly journals A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors

1996 ◽  
Vol 318 (1) ◽  
pp. 241-245 ◽  
Author(s):  
Hedley A COPPOCK ◽  
Ali A OWJI ◽  
Stephen R BLOOM ◽  
David M SMITH
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Binding Site ◽  
Binding Sites ◽  
Calcitonin Gene Related Peptide ◽  
Dependent Protein Kinase ◽  
Related Peptide ◽  
Camp Dependent Protein Kinase ◽  
Calcitonin Gene ◽  
Dependent Protein

We have previously demonstrated specific binding sites for adrenomedullin, a novel member of the calcitonin family of peptides, in rat muscles. It is unclear whether these receptors are vascular or muscular. Receptors for the structurally similar calcitonin gene-related peptide (CGRP) are present on myocytes and might be involved in the regulation of myocyte glucose metabolism and control by motor neurons. We investigated whether adrenomedullin binding sites were present on L6 myocytes. Specific [125I]adrenomedullin binding sites were demonstrated where adrenomedullin competed with an IC50 of 0.22±0.04 nM (mean±S.E.M.) and a concentration of binding sites (Bmax) of 0.95±0.19 pmol/mg of protein (mean±S.E.M.). CGRP and the specific CGRP receptor antagonist CGRP(8–37) competed weakly at this site (IC50 > 10 and 601±298 nM respectively). Binding studies with [125I]CGRP revealed a binding site for CGRP (IC50 = 0.13±0.01 nM; Bmax = 0.83±0.10 pmol/mg of protein) where both CGRP(8–37) and adrenomedullin competed with [125I]CGRP with IC50 values of 1.15±0.12 and 8.68±0.98 nM respectively. Chemical cross-linking showed the CGRP and adrenomedullin binding site–ligand complexes to have approximate molecular masses of 82 and 76 kDa respectively. Both CGRP and adrenomedullin increased adenylate cyclase activity with similar potencies. In both cases adenylate cyclase activation was blocked by CGRP(8–37). Stimulation with 10 nM adrenomedullin or CGRP caused an increase in the percentage of total activated cellular cAMP-dependent protein kinase from 38% in resting cells to 100% and 98% respectively. Therefore in L6 cells adrenomedullin can bind to CGRP receptors, activating adenylate cyclase and cAMP-dependent protein kinase.

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