TY - JOUR
T1 - Selective regulation of Gα(q/11) by an RGS domain in the G protein- coupled receptor kinase, GRK2
AU - Carman, Christopher V.
AU - Parent, Jean Luc
AU - Day, Peter W.
AU - Pronin, Alexey N.
AU - Sternweis, Pamela M.
AU - Wedegaertner, Philip B.
AU - Gilman, Alfred G.
AU - Benovic, Jeffrey L.
AU - Kozasa, Tohru
PY - 1999/11/26
Y1 - 1999/11/26
N2 - G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein α subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF4/--dependent binding to G protein α subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF4/-. This revealed the specific ability of bovine brain Gα(q/11) to bind to both GRK2 and GRK3 in an AlF4/--dependent manner. In contrast, Gα(s), Gα(i), and Gα(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain G2a(q11) could also bind to an N-terminal construct of GRK2, while no binding of Gα(q/11), Gα(s), Gα(i), or Gα(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Gα(q) revealed significant binding of both Gα(q) GDP/AlF4/- and Gα(q)(GTPγS), but not Gα(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Gα(q)(R183C) but not wild type Gα(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Gα(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Gα(q)-mediated activation of phospholipase C-β both in vitro and in cells, possibly through sequestration of activated Gα(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.
AB - G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein α subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF4/--dependent binding to G protein α subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF4/-. This revealed the specific ability of bovine brain Gα(q/11) to bind to both GRK2 and GRK3 in an AlF4/--dependent manner. In contrast, Gα(s), Gα(i), and Gα(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain G2a(q11) could also bind to an N-terminal construct of GRK2, while no binding of Gα(q/11), Gα(s), Gα(i), or Gα(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Gα(q) revealed significant binding of both Gα(q) GDP/AlF4/- and Gα(q)(GTPγS), but not Gα(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Gα(q)(R183C) but not wild type Gα(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Gα(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Gα(q)-mediated activation of phospholipase C-β both in vitro and in cells, possibly through sequestration of activated Gα(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.
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U2 - 10.1074/jbc.274.48.34483
DO - 10.1074/jbc.274.48.34483
M3 - Article
C2 - 10567430
AN - SCOPUS:0033607326
SN - 0021-9258
VL - 274
SP - 34483
EP - 34492
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 48
ER -