The role of calmodulin recruitment in Ca2+ stimulation of adenylyl cyclase type 8.
Ca2+ stimulation of adenylyl cyclase type 8 (AC8) is mediated by calmodulin (CaM). An earlier study identified two CaM binding sites in AC8; one that was apparently not essential for AC8 activity, located at the N terminus, and a second site that was critical for Ca2+ stimulation, found at the C terminus (Gu, C., and Cooper, D. M. F. (1999) J. Biol. Chem. 274, 8012-8021). This study explores the role of these two CaM binding domains and their interaction in regulating AC8 activity, employing binding and functional studies with mutant CaM and modified AC8 species. We report that the N-terminal CaM binding domain of AC8 has a role in recruiting CaM and that this recruitment is essential to permit stimulation by Ca2+ in vivo. Using Ca2+-insensitive mutants of CaM, we found that partially liganded CaM can bind to AC8, but only fully liganded Ca2+/CaM can stimulate AC8 activity. Moreover, partially liganded CaM inhibited AC8 activity in vivo. The results indicate that CaM pre-associates with the N terminus of AC8, and we suggest that this recruited CaM is used by the C terminus of AC8 to mediate Ca2+ stimulation.
Simpson
Rachel E
RE
The Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
Ciruela
Antonio
A
The Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
Cooper
Dermot M F
DMF
The Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom. Electronic address: dmfc2@cam.ac.uk.
D000262:Adenylyl Cyclases / Q000378:metabolism* / Q000502:physiology*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D002147:Calmodulin; D005982:Glutathione Transferase; D000262:Adenylyl Cyclases; C118292:adenylyl cyclase 8; D002118:Calcium