The regulation of type 7 adenylyl cyclase by its C1b region and Escherichia coli peptidylprolyl isomerase, SlyD

Mammalian membrane-bound adenylyl cyclase consists of two highly conserved cytoplasmic domains (C1a and C2a) separated by a less conserved connecting region, C1b, and one of two transmembrane domains, M2, The C1a and C2a doma ins form a catalytic core that can be stimulated by forskolin and the stimu latory G protein subunit alpha (G alpha (s)), In this study, we analyzed th e regulation of type 7 adenylyl cyclase (AC7) by C1b. The C1a, G1b, and C2a domains of AC7 were purified separately. Escherichia coli SlyD protein, a cis-trans peptidylprolyl isomerase (PPIase), copurifies with AC7 C1b (7C1b) , SlyD protein can inhibit the G alpha (s)- and/or forskolin-activated acti vity of both soluble and membrane-bound AC7, Mutant forms of SlyD with redu ced PPIase activity are less potent in the inhibition of AC7 activity. Inte restingly, different isoforms of mammalian membrane-bound adenylyl cyclase can be either inhibited or stimulated by SlyD protein, raising the possibil ity that mammalian PPIase may regulate enzymatic activity of mammalian aden ylyl cyclase. Purified 7C1b-SlyD complex has a greater inhibitory effect on AC7 activity than SlyD alone. This inhibition by 7C1b is abolished in a 7C 1b mutant in which a conserved glutamic acid (amino acid residue 582) is ch anged to alanine, Inhibition of adenylyl cyclase activity by 7C1b is furthe r confirmed by using 7C1b purified from an E. coli slyD-deficient strain. T his inhibitory activity of AC7 is also observed with the 28-mer peptides de rived from a region of C1b conserved in AC7 and AC2 but is not observed wit h a peptide derived from the corresponding region of AC6. This inhibitory a ctivity exhibited by the C1b domain may result from the interaction of 7C1b with 7C1a and 7C2a and may serve to hold AC7 in the basal nonstimulated st ate.