Gβγ Activation Site in Adenylyl Cyclase Type II

Abstract

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...

Regulation of cAMP Responses by the G12/13 Pathway Converges on Adenylyl Cyclase VII

“…AC7 has been grouped together with AC2 and AC4 as a subfamily of AC isozymes based on sequence similarities and their regulation by G protein ␤␥ subunits (11,26,27,38). However, our results suggest that AC7, but not AC2, is a specific AC isoform required for regulation of cAMP by the G 12/13 pathway.…”

“…These two isoforms show over 70% sequence identity in the catalytic domains and are similarly regulated by G␤␥ based on in vitro assays (26,27). As a control we chose to express AC2 in HEK 293 cells for comparison of responses with those seen by expression of AC7.…”

Conditional Stimulation of Type V and VI Adenylyl Cyclases by G Protein βγ Subunits

“…Thus, here onward the term "conditional stimulation" is used to describe the effects of G␤␥ subunits on ACV and ACVI. Because the G␤␥ binding sites on ACII have been reported to reside in the C2 domain and the C1b region (18,19,42), we investigated whether G␤ 1 ␥ 2 regulated the activity of the recombinant, engineered, soluble forms of canine ACV and hACVI that can be activated by G␣ s and forskolin (5, 7). The canine ACV was used because, except for one residue in each of its C1 and C2 domains, the sequences of these regions are identical to those of the corresponding regions in hACV.…”

“…Thus, even the conditional stimulation of the different isoforms by G␤␥ subunits varies among the different AC isoforms. The G␤␥ interaction sites on ACII reside on the C1b and C2 regions (18,19,42), and the sequence (PFAHL) on the C1b region that binds G␤␥ is conserved in ACIV and ACVII (19). Interestingly, we have previously shown that G␤␥ interactions with ACI also involve the C1b and C2 regions of this enzyme (9), although ACI is inhibited by G␤␥ subunits, whereas ACII is stimulated by G␤␥.…”

“…For instance, the C-terminal region of the C1 domain, C1b, is highly variable among the nine AC isoforms and has been found to be involved in some of the type-specific characteristics. In ACII, ACIV, and ACVII, the C1b region is required for stimulatory actions of G␤␥ subunits (18,19). The C1b region in ACI is also necessary for the binding of Ca 2ϩ /calmodulin and consequent stimulation of enzyme activity (20,21) as well as G␤␥-mediated inhibition of activity (9).…”