Abstract
The interaction between the alpha subunit of G protein Gs (Gsalpha) and the two cytoplasmic domains of adenylyl cyclase (C1 and C2) is a key step in the stimulation of cAMP synthesis by hormones. Mutational analysis reveals that three discrete regions in the primary sequence of adenylyl cyclase affect the EC50 values for Gsalpha activation and thus are the affinity determinants of Gsalpha. Based on the three-dimensional structure of C2.forskolin dimer, these three regions (C2 alpha2, C2 alpha3/beta4, and C1 beta1) are close together and form a negatively charged and hydrophobic groove the width of an alpha helix that can accommodate the positively charged adenylyl cyclase binding region of Gsalpha. Two mutations in the C2 alpha3/beta4 region decrease the Vmax values of Gsalpha activation without an increase in the EC50 values. Since these three regions are distal to the catalytic site, the likely mechanism for Gsalpha activation is to modulate the structure of the active site by controlling the orientation of the C2 alpha2 and alpha3/beta4 structures.
Highlights
From the ‡Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637 and the §Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0580
We describe mutations at three discrete regions of the soluble adenylyl cyclase, one in the IC1 protein and two in the IIC2 protein, that significantly affect Gs␣ activation with little change in forskolin activation
We tested for Gs␣ and forskolin activation using E. coli lysates containing the IIC2 mutant proteins and wild type IC1
Summary
Construction, Expression, and Purification of Wild Type and Mutant Forms of IC1 and IIC2 Proteins—Plasmids used to express mutant forms of IC1 and IIC2 were constructed by site-directed mutagenesis using pProExHAH6-IC1 or -IIC2 as the phagemid [9]. To express wild type and mutant forms of hexohistidine-tagged IC1 and IIC2, the plasmids that encoded wild type or mutant forms of IC1 or IIC2 were transformed into Escherichia coli BL21(DE3) cells. The construction of plasmid H6-pQE60-Gs␣ and the expression and purification of hexohistidine-tagged Gs␣ were performed as described [10]. Molecular Modeling of the Interaction between Gs␣ and Mammalian Adenylyl Cyclase—The Gs␣ structure was modeled using the sequence alignment and homology-modeling program LOOK version 2.0 (Molecular Applications Group) based on its sequence homology to GTP␥Sbound forms of bovine G protein transducin ␣ [13]. Gs␣ was docked onto the C1C2 heterodimer using program O [16] and data from the mutational analysis of Gs␣ and C1C2 soluble adenylyl cyclase (Ref. 17 and this paper)
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