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The G␤␥ complex of heterotrimeric G proteins is the most outstanding example for the divergent regulation of mammalian adenylyl cyclases. The heterodimeric G␤␥ complex inhibits some isoforms, e.g. ACI, and stimulates the isoforms ACII, -IV, and -VII. Although former studies identified the QEHA region located in the C 2 domain of ACII as an important interaction site for G␤␥, the determinant of the stimulatory effect of G␤␥ has not been detected. Here, we identified the C 1b domain as the stimulatory region using full-length adenylyl cyclase. The relevant G␤␥ signal transfer motif in IIC 1b was determined as MTRYLESWGAAKPFAHL (amino acids 493-509). Amino acids of this PFAHL motif were absolutely necessary for ACII to be stimulated by G␤␥, whereas they were dispensable for G␣ s or forskolin stimulation. The PFAHL motif is present in all three adenylyl cyclase isoforms that are activated by G␤␥ but is absent in other adenylyl cyclase isoforms as well as other known effectors of G␤␥. The emerging concept of two contact sites on different molecule halves for effective regulation of adenylyl cyclase is discussed.Adenylyl cyclase (AC), 2 the enzyme that synthesizes the universal second messenger cAMP, is a key player in intracellular signaling pathways of hormones, neurotransmitters, odorants, and chemokines. It is subject to coincident regulation by extracellular stimuli. Particulate mammalian ACs are represented by at least nine different isoforms (ACI-ACIX) that have been cloned and analyzed (1). All isoforms can be activated by the ␣ subunit of the heterotrimeric stimulatory guanine nucleotide-binding protein (G␣ s ) and with the exception of ACIX, also by the diterpene forskolin.ACs are integral membrane proteins with a common topology (2) consisting of two sets of six transmembrane spans (M 1 and M 2 ) each followed by a cytosolic domain (C 1 and C 2 ). Both cytosolic domains can be subdivided on the basis of sequence similarity in domains C a and C b . The C 1a domain equals C 2a in ϳ60% of amino acids, and both subdomains heterodimerize to form the pseudosymmetrical catalytic core. They can be expressed as independent polypeptides and when mixed form a heterodimer that exhibits catalytic activity as well as sensitivity to G␣ s and forskolin (3, 4). Outside of the catalytic core primary sequences significantly differ between the individual AC isoforms. The mechanisms of catalysis and activation by G␣ s were deduced from crystal structures of the soluble catalytic core bound to various regulators (5-7). However, little is known about the molecular mechanisms of isoform-specific regulation of ACs, i.e. by regulators other than G␣ s .Known regulators of ACs include Ca 2ϩ ions, Ca 2ϩ /calmodulin, Ca 2ϩ / calcineurin, cAMP-dependent protein kinase, Ca 2ϩ -dependent protein kinase, the ␣ subunits of G i , G o , G z , and the G protein ␤␥ complex (8). G␤␥ is a conditional regulator, i.e. both activation and inhibition are best observed at the prestimulated AC. G␤␥ has opposing effects on different subtypes of AC; it ei...

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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