Carmen W. Dessauer

Interaction of Gsalpha with the cytosolic domains of mammalian adenylyl cyclase.

Forskolin- and Gsα-stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized ∼25-kDa cytosolic fragments derived from mammalian adenylyl cyclases (native M r ∼ 120,000). The C1a domain from type V adenylyl cyclase (VC1) and the C2 domain from type II adenylyl cyclase (IIC2) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity, 150 μmol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity complex containing one molecule each of VC1, IIC2, and guanosine 5′-O-(3-thiotriphosphate) (GTPγS)-Gsα is responsible for the observed enzymatic activity and can be isolated. In addition, GTPγS-Gsαinteracts with homodimers of IIC2 to form a heterodimeric complex (one molecule each of Gsα and IIC2) but not detectably with homodimers of VC1. Nevertheless, Gsα can be cross-linked to VC1 in the activated heterotrimeric complex of VC1, IIC2, and Gsα, indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272–22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site at the interface between C1 and C2.

Identification of a Gialpha binding site on type V adenylyl cyclase.

The stimulatory G protein α subunit Gsα binds within a cleft in adenylyl cyclase formed by the α1-α2 and α3-β4 loops of the C2 domain. The pseudosymmetry of the C1 and C2 domains of adenylyl cyclase suggests that the homologous inhibitory α subunit Giα could bind to the analogous cleft within C1. We demonstrate that myristoylated guanosine 5′-3-O-(thio)triphosphate-Giα1 forms a stable complex with the C1 (but not the C2) domain of type V adenylyl cyclase. Mutagenesis of the membrane-bound enzyme identified residues whose alteration either increased or substantially decreased the IC50 for inhibition by Giα1. These mutations suggest binding of Giα within the cleft formed by the α2 and α3 helices of C1, analogous to the Gsα binding site in C2. Adenylyl cyclase activity reconstituted by mixture of the C1 and C2 domains of type V adenylyl cyclase was also inhibited by Giα. The C1b domain of the type V enzyme contributed to affinity for Giα, but the source of C2 had little effect. Mutations in this soluble system faithfully reflected the phenotypes observed with the membrane-bound enzyme. The pseudosymmetrical structure of adenylyl cyclase permits bidirectional regulation of activity by homologous G protein α subunits.

The Catalytic Mechanism of Mammalian Adenylyl Cyclase EQUILIBRIUM BINDING AND KINETIC ANALYSIS OF P-SITE INHIBITION

The mechanism of P-site inhibition of adenylyl cyclase has been probed by equilibrium binding measurements using 2′-[3H]deoxyadenosine, a P-site inhibitor, and by kinetic analysis of both the forward and reverse reactions (i.e. cyclic AMP and ATP synthesis, respectively). There is one binding site for 2′-deoxyadenosine per C1/C2 heterodimer; the K d is 40 ± 3 μm. Binding is observed only in the presence of one of the products of the adenylyl cyclase reaction, pyrophosphate (PPi). A substrate analog, Ap(CH2)pp (α,β-methylene adenosine 5′-triphosphate), and cyclic AMP compete for the P-site in the presence of PPi, but P-site analogs do not compete for substrate binding (in the absence of PPi). Kinetic analysis indicates that release of products from the enzyme is random. These facts permit formulation of a model for the adenylyl cyclase reaction, for which we provide substantial kinetic support. We propose that P-site analogs act as dead-end inhibitors of product release, stabilizing an enzyme-product (E-PPi) complex by binding at the active site. Although product release is random, cyclic AMP dissociates from the enzyme preferentially. Release of PPiis slow and partially rate-limiting.

Interactions of forskolin and ATP with the cytosolic domains of mammalian adenylyl cyclase

Fragments of the two cytoplasmic domains of mammalian adenylyl cyclases can be synthesized independently (and abundantly) as soluble proteins; Gsα- and forskolin-stimulated enzymatic activity is restored upon their mixture. We have utilized this system to characterize the interactions of adenylyl cyclase with forskolin and its substrate, ATP. In the presence of Gsα, adenylyl cyclase is activated in response to occupation of only one forskolin-binding site. A single binding site for forskolin was identified by equilibrium dialysis; itsK d (0.1 μm) corresponds to the EC50 for enzyme activation. The affinity of forskolin for adenylyl cyclase is greatly reduced in the absence of Gsα(∼40 μm). Binding of forskolin to the individual cytoplasmic domains of the enzyme was not detected. A single binding site for the ATP analog, α,β-methylene ATP (Ap(CH2)pp), was also detected by equilibrium dialysis. Such binding was not observed with the individual domains. Binding of Ap(CH2)pp was unaffected by P-site inhibitors of adenylyl cyclase. A modified P-loop sequence located near the carboxyl terminus of adenylyl cyclase has been implicated in ATP binding. Mutation of the conserved, non-glycine residues within this region caused no significant changes in the K m for ATP or the K i for Ap(CH2)pp. It thus seems unlikely that this region is part of the active site. However, a mutation in the C1 domain (E518A) causes a 10-fold decrease in the binding affinity for Ap(CH2)pp. This residue and the active site of the enzyme may lie at the interface between the two cytosolic domains.

The interactions of adenylate cyclases with P-site inhibitors.

Recent kinetic, binding and crystallographic studies using P-site inhibitors of mammalian adenylate bases provide new insights into the catalytic mechanism of these highly regulated enzymes. Here, Carmen Dessauer and colleagues discuss the conformational states of adenylate cyclase, the structural determinants of inhibitor binding and the potential uses of these inhibitors as pharmacological agents.

Purification and characterization of a soluble form of mammalian adenylyl cyclase

An engineered, soluble form of mammalian adenylyl cyclase has been expressed in Escherichia coli and purified by three chromatographic steps. The enzyme utilizes one molecule of ATP to synthesize one molecule of cyclic AMP and pyrophosphate at a maximal specific activity of 12.8 μmol/min/mg, corresponding to a turnover number of 720 min−1. Although devoid of membrane spans, the enzyme displays all of the regulatory properties that are common to mammalian adenylyl cyclases. It is activated synergistically by Gsα and forskolin and is inhibited by adenosine (P-site) analogs with kinetic patterns that are identical to those displayed by the native enzymes. The purified enzyme is also inhibited directly by the G protein βγ subunit complex. After adenovirus-mediated expression in adenylyl cyclase-deficient HC-1 cells, the enzyme can be stimulated synergistically by Gs-coupled receptors and forskolin.

Regulation of L‐type Ca2+ channels in rabbit portal vein by G protein αs and βγ subunits

1 The effect of purified G protein subunits αs and βγ on L‐type Ca2+ channels in vascular smooth muscle and the possible pathways involved were investigated using freshly isolated smooth muscle cells from rabbit portal vein and the whole‐cell patch clamp technique. 2 Cells dialysed with either Gαs or Gβγ exhibited significant increases in peak Ba2+ current (IBa) density (148 % and 131 %, respectively) compared with control cells. The combination of Gαs and Gβγ further increased peak IBa density (181 %). Inactive Gαs and Gβγ did not have any effect on Ca2+ channels. 3 The stimulatory effect of Gαs on peak IBa was entirely abolished by the protein kinase A inhibitor Rp‐8‐Br‐cAMPS, or the adenylyl cyclase inhibitor SQ 22536. On the other hand, the stimulatory response of Ca2+ channels to Gβγ was not affected by the protein kinase A inhibitors Rp‐8‐Br‐cAMPS and KT 5720, or by the Ca2+‐dependent protein kinase C inhibitor bisindolylmaleimide 1, but was completely blocked by the protein kinase C inhibitor calphostin C. Pretreatment of cells with phorbol 12‐myristate 13‐acetate for over 18 h prevented the stimulatory effect of Gβγ on peak IBa. In addition, acute application of phorbol 12,13‐dibutyrate enhanced peak IBa density in control cells, which could be entirely blocked by calphostin C. 4 These data indicate that enhancement of Ba2+ currents by Gαs and Gβγ can be attributed to increased activity of protein kinase A and protein kinase C, respectively. No direct membrane‐delimited pathway for Ca2+ channel regulation by activated Gs proteins could be detected in vascular smooth muscle cells.