Mammalian ACs (adenylyl cyclases) are integrating effector molecules in signal transduction regulated by a plethora of molecules in either an additive, synergistic or antagonistic manner. Out of nine different isoforms, each AC subtype uses an individual set of regulators. In the present study, we have used chimaeric constructs, point mutations and peptide competition studies with ACs to show a common mechanism of multiple contact sites for the regulatory molecules Gβγ and calmodulin. Despite their chemical, structural and functional variety and different target motifs on AC, Gβγ and calmodulin share a two-site-interaction mechanism with Gαs and forskolin to modulate AC activity. Forskolin and Gαs are known to interact with both cytosolic domains of AC, from inside the catalytic cleft as well as at the periphery. An individual interaction site located at C1 of the specifically regulated AC subtype had been ascribed for both Gβγ and calmodulin. In the present study we now show for these two regulators of AC that a second isoform- and regulator-specific contact site in C2 is necessary to render enzyme activity susceptible to Gβγ or calmodulin modulation. In addition to the PFAHL motif in C1b of ACII, Gβγ contacts the KF loop in C2, whereas calmodulin requires not only the Ca2+-independent AC28 region in C1b but also a Ca2+-dependent domain in C2a of ACI containing the VLG loop to stimulate this AC isoform.

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