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J. Biol. Chem., Vol. 275, Issue 37, 28500-28506, September 15, 2000

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Polarity Exchange at the Interface of Regulators of G Protein Signaling with G Protein -Subunits^*

Thomas Wieland^§, Nehat Bahtijari, Xiao-Bo Zhou, Christiane Kleuss^¶, and Melvin I. Simon

From the  Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Universitäts-Krankenhaus Eppendorf, Martinistrasse 52, D-20246 Hamburg, the ^¶ Institut für Pharmakologie, Freie Universität Berlin, Thielallee 67-73, D-14195 Berlin, Germany, and the  Division of Biology, California Institute of Technology, Pasadena, California 91125

RGS proteins are GTPase-activating proteins (GAPs) for G protein -subunits. This GAP activity is mediated by the interaction of conserved residues on regulator of G protein signaling (RGS) proteins and G-subunits. We mutated the important contact sites Glu-89, Asn-90, and Asn-130 in RGS16 to lysine, aspartate, and alanine, respectively. The interaction of RGS16 and its mutants with G_t and G_i1 was studied. The GAP activities of RGS16N90D and RGS16N130A were strongly attenuated. RGS16E89K increased GTP hydrolysis of G_i1 by a similar extent, but with an about 100-fold reduced affinity compared with non-mutated RGS16. As Glu-89 in RGS16 is interacting with Lys-210 in G_i1, this lysine was changed to glutamate for compensation. G_i1K210E was insensitive to RGS16 but interacted with RGS16E89K. In rat uterine smooth muscle cells, wild type RGS16 abolished G_i-mediated ₂-adrenoreceptor signaling, whereas RGS16E89K was without effect. Both G_i1 and G_i1K210E mimicked the effect of ₂-adrenoreceptor stimulation. G_i1K210E was sensitive to RGS16E89K and 10-fold more potent than G_i1. Analogous mutants of G_q (G_qK215E) and RGS4 (RGS4E87K) were created and studied in COS-7 cells. The activity of wild type G_q was counteracted by wild type RGS4 but not by RGS4E87K. The activity of G_qK215E was inhibited by RGS4E87K, whereas non-mutated RGS4 was ineffective. We conclude that mutation of a conserved lysine residue to glutamate in G_i and G_q family members renders these proteins insensitive to wild type RGS proteins. Nevertheless, they are sensitive to glutamate to lysine mutants of RGS proteins. Such mutant pairs will be helpful tools in analyzing G-RGS specificities in living cells.

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