title: Distinct mechanisms of calmodulin binding and regulation of adenylyl cyclases 1 and 8.
abstract: Calmodulin (CaM), by mediating the stimulation of the activity of two adenylyl cyclases (ACs), plays a key role in integrating the cAMP and Ca(2+) signaling systems. These ACs, AC1 and AC8, by decoding discrete Ca(2+) signals can contribute to fine-tuning intracellular cAMP dynamics, particularly in neurons where they predominate. CaM comprises an α-helical linker separating two globular regions at the N-terminus and the C-terminus that each bind two Ca(2+) ions. These two lobes have differing affinities for Ca(2+), and they can interact with target proteins independently. This study explores previous indications that the two lobes of CaM can regulate AC1 and AC8 differently and thereby yield different responses to cellular transitions in [Ca(2+)](i). We first compared by glutathione S-transferase pull-down assays and offline nanoelectrospray ionization mass spectrometry the interaction of CaM and Ca(2+)-binding deficient mutants of CaM with the internal CaM binding domain (CaMBD) of AC1 and the two terminal CaMBDs of AC8. We then examined the influence of these three CaMBDs on Ca(2+) binding by native and mutated CaM in stopped-flow experiments to quantify their interactions. The three CaMBDs show quite distinct interactions with the two lobes of CaM. These findings establish the critical kinetic differences between the mechanisms of Ca(2+)-CaM activation of AC1 and AC8, which may underpin their different physiological roles.
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
authors.forename: Nanako
authors.initials: N
authors.lastname: Masada
authors.forename: Sabine
authors.initials: S
authors.lastname: Schaks
authors.forename: Sophie E
authors.initials: SE
authors.lastname: Jackson
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
authors.forename: Dermot M F
authors.initials: DM
authors.lastname: Cooper
mesh_terms: D000262:Adenylyl Cyclases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005982:Glutathione Transferase / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D002147:Calmodulin; D005982:Glutathione Transferase; D000262:Adenylyl Cyclases; C106670:adenylyl cyclase 1; C118292:adenylyl cyclase 8; D002118:Calcium