Isolation and Characterization of Constitutively Active Mutants of Mammalian Adenylyl Cyclase

Abstract

A genetic screen in Saccharomyces cerevisiae identified mutations in mammalian adenylyl cyclase that activate the enzyme in the absence of G s ␣. Thirteen of these mutant proteins were characterized biochemically in an assay system that depends on a mixture of the two cytosolic domains (C 1 and C 2 ) of mammalian adenylyl cyclases. Three mutations, I1010M, K1014N, and P1015Q located in the ␤4-␤5 loop of the C 2 domain of type II adenylyl cyclase, increase enzymatic activity in the absence of activators. The K1014N mutation displays both increased maximal activity and apparent affinity for the C 1 domain of type V adenylyl cyclase in the absence of activators of the enzyme. The increased affinity of the mutant C 2 domain of adenylyl cyclase for the wild type C 1 domain was exploited to isolate a complex containing VC 1 , IIC 2 , and G s ␣-guanosine 5-3-O-(thio)triphosphate (GTP␥S) in the absence of forskolin and a complex of VC 1 , IIC 2 , forskolin, and P-site inhibitor in the absence of G s ␣-GTP␥S. The isolation of these complexes should facilitate solution of crystal structures of low activity states of adenylyl cyclase and thus determination of the mechanism of activation of the enzyme by forskolin and G s ␣.Mammalian adenylyl cyclases are membrane-bound enzymes that catalyze the synthesis of the intracellular second messenger cyclic AMP from ATP. Nine isoforms of the enzyme have been detected, and they display characteristic regulatory properties and patterns of cellular distribution (1, 2). Cellular rates of cyclic AMP synthesis are controlled by a variety of extracellular ligands that interact with heptahelical receptors in the plasma membrane. Relevant receptors can either stimulate cyclic AMP synthesis, usually via the intermediacy of a G protein 1 (G s ) that activates adenylyl cyclase, or inhibit cyclic AMP synthesis, often by interaction with an inhibitory G protein, G i . Mammalian adenylyl cyclases can also be activated by the diterpene forskolin (3) and inhibited by certain adenosine analogs and adenine nucleotides called P-site inhibitors (4). Certain adenylyl cyclases are also regulated by Ca 2ϩ , Ca 2ϩ -calmodulin, and phosphorylation (5).Mammalian adenylyl cyclases are integral membrane proteins that appear to contain two sets of six membrane-spanning helices that are separated by a large (ϳ40 kDa) cytoplasmic loop and followed by a similarly sized carboxyl-terminal cytosolic domain (6). The cytosolic domains, termed C 1 and C 2 , have been extensively studied; they are responsible for catalytic activity and most of the regulatory properties of the enzymes (7). The first 200 -250 amino acids of each cytosolic domain, designated C 1a and C 2a , are the most highly conserved regions among adenylyl cyclases. Strikingly, the C 1a and C 2a domains are approximately 50% similar and 25% identical to each other within a single isoform of adenylyl cyclase, and they are 20 -25% similar to the catalytic domains of membrane-bound and cytosolic guanylyl cyclases.The C 1a and C 2a domains of adenylyl cyclas...

Mutations That Rescue the Paralysis of Caenorhabditis elegans ric-8 (Synembryn) Mutants Activate the Gαs Pathway and Define a Third Major Branch of the Synaptic Signaling Network

“…Similarly, we found that the found to increase the affinity of the two domains for gsa-1 strong gain-of-function mutation could not bypass each other (Hatley et al 2000). We note that the acy-1 the larval arrest phenotype of acy-1 nulls, although the (md1756) mutation occurs at a known contact point gsa-1(ce81); acy-1(pk1279) double mutants, like acy-1 between the two domains, as revealed by structural stud-(pk1279) single mutants, can survive for days after hatchies of vertebrate adenylyl cyclases (Tesmer et al 1997;ing (Reynolds et al 2005).…”

Regulation of Adenylyl Cyclases by a Region Outside the Minimally Functional Cytoplasmic Domains

“…Finally, the maximal stimulated activity is ϳ10-fold higher for the soluble enzyme (21,22). Moreover, the F400Y mutation in the type V molecule, which showed increased basal activity and sensitivity to G s ␣ and forskolin, did not alter the enzymatic activity of the soluble construct bearing the same substitution (24,25). It has been shown that the C1b domain of adenylyl cyclases (which is absent in these constructs) possesses regulatory properties (26 -31).…”

“…Hatley et al (25) isolated mutants of the type II enzyme that rescued the cyclase-null Saccharomyces cerevisiae strain. All mutations mapped to the cytoplasmic loops of the enzyme.…”

Regulation and cell biology of secondary metabolite production in Fusarium graminearum and Fusarium pseudograminearum

“…Similarly, Hatley et al (2000) created constitutively active mammalian adenylyl cyclase mutants through single amino acid substitutions, which increased the interaction of the two cytosolic domains of the adenylyl cyclase responsible for the catalytic activity (Tang and Gilman, 1995). One of those mutants, namely P1015Q has a similar amino acid change to our FAC1 P1441S gain-of-function allele, from a rigid proline to a non-polar glutamine (Hatley et al, 2000). Thus the change from proline to a non-polar serine in FAC1 P1441S could potentially cause similar structural changes, which increase the interaction between the substrate binding domain and the catalytic domain.…”

“…In a constitutively active mutant of the Dictyostelium adenylyl cyclase, a mutation in the first cytoplasmic loop was thought to bring this domain closer to the second cytoplasmic loop, which might mimic activation by G proteins (Parent and Devreotes, 1996). Similarly, Hatley et al (2000) created constitutively active mammalian adenylyl cyclase mutants through single amino acid substitutions, which increased the interaction of the two cytosolic domains of the adenylyl cyclase responsible for the catalytic activity (Tang and Gilman, 1995). One of those mutants, namely P1015Q has a similar amino acid change to our FAC1 P1441S gain-of-function allele, from a rigid proline to a non-polar glutamine (Hatley et al, 2000).…”