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All reagents for microinjection were dissolved in microinjection buffer containing 5 mM sodium phosphate (pH 7

All reagents for microinjection were dissolved in microinjection buffer containing 5 mM sodium phosphate (pH 7.2) and 100 mM KCl. or kinase-deficient GRK2 inhibited insulin-stimulated GLUT4 translocation as well as 2-deoxyglucose uptake. Importantly, a mutant GRK2 lacking the RGS domain was without effect. Taken together, these results indicate that through its RGS domain endogenous GRK2 functions as a negative regulator of insulin-stimulated glucose transport by interfering with Gq/11 signaling to GLUT4 translocation. Furthermore, inhibitors of GRK2 can lead to enhanced insulin sensitivity. (2000) and Carman (1999) have reported that GRK2, but not the other subtypes of GRKs, specifically inhibits the activity of Gq/11, but not Gi or Gs. Recent studies have shown that certain signaling proteins, which classically function in GPCR signaling pathways, can also participate in receptor tyrosine kinase (RTK) signaling cascades. For example, IGF-1-mediated MAP kinase phosphorylation is dependent on Gi/ signaling (Luttrell have shown that -arrestin-1 is required for IGF-1-mediated MAP kinase signaling (Lin (1994) have found that inhibition of Gi with pertussis toxin blocks insulin-stimulated phosphatidylinositol-glycan hydrolysis, phosphatidic acid synthesis, and diacylglycerol production, but had no effect on insulin-stimulated glucose transport. This latter finding is consistent with other reports showing no effect of pertussis toxin on insulin-stimulated glucose transport or GLUT4 translocation (Ploug em et al /em , 1997; Imamura em et al /em , 1999a). On the other hand, it has been shown that genetic deletion of Gi leads to a state of insulin resistance in mice (Moxham and Malbon, 1996), whereas transgenic expression of a constitutively active SR9238 Gi (Q205L) leads to enhanced insulin stimulation of glucose transport and GLUT4 translocation (Chen em et al /em , 1997), and this may be mediated by the effect of Q205L to inhibit PTP1B SR9238 activity (Tao em et al /em , 2001). The 2-adrenergic receptor (2AR) is a GPCR that can interact with the insulin signaling system. Thus, acute insulin treatment enhances ligand-mediated internalization of the 2AR, SR9238 and reduces cAMP generated following treatment with 2AR ligands (Baltensperger em et al /em , 1996). Insulin treatment leads to phosphorylation of 2AR Tyr350 creating an SH2 domain binding site that mediates Grb2 association and is required for both insulin-induced 2AR internalization and counter-regulation of cAMP generation (Karoor em et al /em , 1998). The 2AR also contains a consensus sequence for Akt, and insulin-induced Akt phosphorylation of Ser345 and Ser346 is also required for 2AR internalization following insulin treatment (Doronin em et al /em , 2002). In addition, chronic adrenergic stimulation can counter-regulate insulin action leading to a state of insulin resistance (Deibert and DeFronzo, 1980), and it is possible that this could be, at least in part, mediated through GRK2. Thus, 2AR activation leads to recruitment of GRK2 to the plasma membrane, and this might facilitate GRK2-induced inhibition of insulin signaling through Gq/11. In summary, these studies demonstrate a novel role for GRK2 as an endogenous protein inhibitor of the insulin signaling pathway leading to glucose transport stimulation. The data are consistent with the view that GRK2 performs this function by RGS domain-mediated inhibition of the Gq/11 branch of the insulin/glucose transport stimulatory pathway. Since inhibition of endogenous GRK2 leads to cellular insulin sensitization, these results also raise the possibility that GRK2 may be an important target for antidiabetic therapeutics. Chemical inhibitors of GRK2 would be expected to act as insulin sensitizers, which could have beneficial effects in a wide variety of insulin-resistant human conditions, including type II diabetes mellitus. Materials and methods Materials Mouse monoclonal anti-cdc42 antibody, rabbit polyclonal anti-p85 (N-SH2) and anti-IRS-1 antibodies, cdc42 assay kit and protein A agarose were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Mouse monoclonal anti-phosphotyrosine (PY20) antibody was from Transduction Laboratories (Lexington, KY). Rabbit polyclonal anti-GLUT4 antibody was purchased from Chemicon International Inc. (Temecula, CA). Rabbit polyclonal anti-GRK2, anti-GRK3, anti-GRK5, anti-GRK6, ATF3 anti-Gq/11, and anti-cdc42 (P1) antibodies, and horseradish peroxidase-linked anti-rabbit and anti-mouse antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Sheep IgG and fluorescein isothiocyanate (FITC)-conjugated and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated anti-rabbit and anti-mouse IgG antibodies were from Jackson Immunoresearch Laboratories Inc. (West Grove, PA). SuperFECT was purchased from Qiagen (Valencia, CA). Oligofectamine was purchased from Invitrogen (Carlsbad, CA). SiRNA of GRK2 (sense: UGA CUU CAG UGU GCA UCG A dAdT; antisense: U CGA UGC ACA CUG AAG UCA dAdT) was purchased from Dharmacon (Lafayette, CO). Dulbecco’s modified Eagle’s medium (DMEM), Opti-MEM I, and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Grand Island, NY). Plasmid vectors encoding WT- and SR9238 KD-(K220R) GRK2 were kindly provided by Dr Robert J Lefkowitz (Duke University, NC). All radioisotopes were from ICN (Costa Mesa, CA). All other reagents were purchased from Sigma Chemical Co. (St Louis, MO). SR9238 Construction of deletion mutant of GRK2 A deletion mutant of.