Elsevier

Methods in Enzymology

Volume 345, 2002, Pages 127-140
Methods in Enzymology

[10] - Expression, Purification, and Assay of Cytosolic (Catalytic) Domains of Membrane-Bound Mammalian Adenylyl Cyclases

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Introduction

Hormonal regulation of intracellular concentrations of cyclic AMP is exerted primarily by modulation of the catalytic activities of the membrane-bound forms of adenylyl cyclase. To date, nine isoforms of the membrane-bound mammalian enzyme and one cytosolic protein have been identified.1, 2, 3 All membrane-bound mammalian adenylyl cyclases are activated by the α subunit of the stimulatory G protein, Gs. With the exception of type IX, all forms are also activated by forskolin, a diterpene. Only types I, V, and VI are inhibited by the α subunit of the inhibitory G protein Gi, but all isoforms are inhibited by a group of adenosine analogs called P-site inhibitors.1,4

Membrane-bound adenylyl cyclases are composed of two transmembrane domains (each likely containing six membrane-spanning helices) separated by a large cytosolic loop (C1 domain). In addition, the proteins contain a second cytosolic domain at their carboxyl terminus (C2 domain). This topological arrangement is reminiscent of those of the multidrug resistance transporters and the cystic fibrosis conductance regulators.5 Each of the cytosolic domains has similar amino acid sequences (approximately 200 residues), and these sequences are also conserved among both mammalian adenylyl cyclases and guanylyl cyclases. Indeed, it is these sequences that represent the catalytic site of the purine nucleoside triphosphate cyclases.6

After expression in bacteria, the two cytosolic domains of adenylyl cyclase together display most of the properties of the membrane-bound parent molecules, including the capacity to be regulated by G protein α subunits, inhibited by P-site inhibitors, and activated by forskolin.7, 8, 9, 10, 11 The atomic structure of the catalytic domain of adenylyl cyclase bound to activators Gsα and forskolin has been determined.12,13 Structures of adenylyl cyclase bound with various substrate and product inhibitors have also been delineated.14 Together with the results of site-directed mutagenesis and kinetic analysis, these structures have provided detailed insight into the catalytic mechanism of adenylyl cyclase.14,15 Notably, two metal ions are required for cyclic AMP synthesis, similar to the reactions catalyzed by DNA polymerases, reverse transcriptases, and RNA spliceosomes.16

This article describes the construction of bacterial expression vectors encoding the cytosolic domains of adenylyl cyclase, the methods for expression and purification of these proteins, and the conditions for assessing their collaborative catalytic activity.

Section snippets

Construction of Expression Vectors

Tang and Gilman6 first noted that the isolated cytoplasmic domains of adenylyl cyclase displayed regulated catalytic activity. They described the construction, expression, and characterization of a linked chimera between the C1 domain of type I adenylyl cyclase and the C2 domain of the type II enzyme. The molecule was subsequently purified and extensively characterized,17 revealing properties that were remarkably similar to those of the purified, detergent-extracted parent form of the enzyme.18

Expression of Adenylyl Cyclase Cytosolic Domains

Each of the two cytosolic domains of mammalian adenylyl cyclase is expressed in E. coli separately and purified to homogeneity. The bacterial strain BL21(DE3) is cotransformed with one of the expression plasmids encoding a cyclase domain (described above) and pREP4 (Qiagen). The pREP4 plasmid allows constitutive expression of lacI repressor under kanamycin selection. These colonies are used to inoculate 100 ml of Luria broth (LB) containing a 50-μg/ml concentration of both ampicillin and

Assays of Soluble Domains

The activity of adenylyl cyclase is determined by measuring the conversion of [α-32P]ATP to [32P]cAMP. The assay is initiated by combining the labeled substrate with the mixed soluble protein domains and terminated by addition of sodium dodecyl sulfate [2% (w/v), final] and unlabeled cAMP (1.75 mM, final) and ATP (50 mM, final). The procedure for isolation of [32P]cAMP is identical to that used to assay the membrane-bound form of adenylyl cyclase.23,24 In general, no ATP-regenerating system is

Summary

The identification and isolation of the soluble catalytic domains of adenylyl cyclase have provided investigators with useful reagents for the study of these enzymes. They have permitted detailed mechanistic investigation of the actions of forskolin, Gsα, and the inhibitory G protein, Giα.11 They have served as critical reagents for the development of plausible models of the catalytic mechanism of the enzyme. They have enabled X-ray crystallographic analysis of adenylyl cyclase; this technique

Acknowledgments

We thank Michelle Clark and Julie Collins for excellent technical assistance. This work was supported by grants from the Medical Research Council of Canada (to R.K.S.) and the National Institute of General Medical Sciences (GM34497 to A.G.G.) and by the Raymond and Ellen Willie Distinguished Chair in Molecular Neuropharmacology (A.G.G.).

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