Altered Expression of Gq/11 Protein Shapes mGlu1 and mGlu5 Receptor-Mediated Single Cell Inositol 1,4,5- Trisphosphate and Ca Signaling

The metabotropic glutamate (mGlu) receptors mGlu1 and mGlu5 mediate distinct inositol 1,4,5-trisphosphate (IP3) and Ca signaling patterns, governed in part by differential mechanisms of feedback regulation after activation. Single cell imaging has shown that mGlu1 receptors initiate sustained elevations in IP3 and Ca 2 , which are sensitive to agonist concentration. In contrast, mGlu5 receptors are subject to cyclical PKC-dependent uncoupling and consequently mediate coincident IP3 and Ca 2 oscillations that are largely independent of agonist concentration. In this study, we investigated the contribution of Gq/11 protein expression levels in shaping mGlu1/5 receptor-mediated IP3 and Ca 2 signals, using RNA interference (RNAi). RNAi-mediated knockdown of Gq/11 almost abolished the single-cell increase in IP3 caused by mGlu1 and mGlu5 receptor activation. For the mGlu1 receptor, this unmasked baseline Ca oscillations that persisted even at maximal agonist concentrations. mGlu5 receptor-activated Ca oscillations were still observed but were only initiated at high agonist concentrations. Recombinant overexpression of Gq enhanced IP3 signals after mGlu1 and mGlu5 receptor activation. It is noteworthy that although mGlu5 receptor-mediated IP3 and Ca 2 oscillations in control cells were largely insensitive to agonist concentration, increasing Gq expression converted these oscillatory signatures to sustained plateau responses in a high proportion of cells. In addition to modulating temporal Ca signals, upor down-regulation of Gq/11 expression alters the threshold for the concentration of glutamate at which a measurable Ca signal could be detected. These experiments indicate that altering Gq/11 expression levels differentially affects spatiotemporal aspects of IP3 and Ca 2 signaling mediated by the mGlu1 and mGlu5 receptors. Activation of the phospholipase C (PLC) pathway via coupling of G protein-coupled receptors (GPCRs) to G proteins of the Gq/11 family results in inositol 1,4,5-trisphosphate (IP3) production and mobilization of intracellular calcium (Ca i). Receptor activation can initiate spatially and temporally unique Ca signals and thereby regulate an array of cellular processes (Berridge et al., 2000). In this study, we have investigated the contribution of Gq/11 protein expression in shaping receptor-initiated IP3 and Ca 2 signaling patterns. The group I metabotropic glutamate receptors mGlu1a and mGlu5a are family C GPCRs. mGlu1a and mGlu5a receptors couple preferentially to the major isoforms of the Gq family, Gq and G11 , and are capable of activating distinct patterns of Ca mobilization (Hermans and Challiss, 2001). Stimulation of the mGlu1a receptor predominantly initiates a peak-and-plateau Ca response, whereas mGlu5a receptor activation mediates a Ca oscillatory response over a broad range of stimulus strengths (Nash et al., 2002). Previous studies proposed that mGlu5 receptor-mediated Ca oscillations occur because of feedback inhibition via PKC-dependent phosphorylation of a single threonine residue (Thr; Kawabata et al., 1996); however, very recent studies have shown that Thr is not phosphorylated but instead plays a This work was supported by the Wellcome Trust of Great Britain (grant 062495) and by a joint Biotechnology and Biological Sciences Research Council and GlaxoSmithKline Ph.D. studentship (to P.J.A.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.014258. ABBREVIATIONS: PLC, phospholipase C; GPCR, G protein-coupled receptor; IP3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; eGFP, enhanced green fluorescent protein; eGFP-PHPLC , pleckstrin homology domain of PLC 1 tagged with enhanced green fluorescent protein; CICR, Ca -induced Ca -release; mGlu, metabotropic glutamate; mACh, muscarinic acetylcholine; RNAi, RNA interference; CHO, Chinese hamster ovary; HEK, human embryonic kidney; siRNA, small interfering RNA; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RT, reverse transcription; KHB, Krebs-Henseleit buffer; RFU, relative fluorescent units; AM, acetoxymethyl ester; RGS, regulator of G protein signaling. 0026-895X/06/6901-174–184$20.00 MOLECULAR PHARMACOLOGY Vol. 69, No. 1 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 14258/3073294 Mol Pharmacol 69:174–184, 2006 Printed in U.S.A. 174 at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from structural/permissive role for the phosphorylation of an adjacent residue (Ser) by PKC (Kim et al., 2005). Similar PKC-dependent Ca oscillations have also been described after glutamate activation of astrocytes (Codazzi et al., 2001) and activation of another family C GPCR, the Ca -sensing receptor (Young et al., 2002). Use of the pleckstrin homology domain of phospholipase C tagged with enhanced green fluorescent protein (eGFP-PHPLC ) has enabled IP3 oscillations underlying mGlu5a receptor-activated Ca oscillations to be observed (Nash et al., 2001, 2002; Nahorski et al., 2003). These PKC-dependent Ca oscillations (referred to as “dynamic uncoupling”) are distinct from regenerative Ca induced Ca -release (CICR), which is generated through an intrinsic property of the IP3 receptor (Thomas et al., 1996; Taylor and Thorn, 2001). CICR oscillations can be maintained with a relatively low steady-state increase in IP3, as observed after activation of the M3 muscarinic acetylcholine (mACh) receptor with a low agonist concentration in the same cell background (Nash et al., 2001). Our previous studies exploring the determinants of mGlu5 receptor signaling led us to propose a model in which Ca oscillation frequency is dependent on receptor expression levels but is largely independent of agonist concentration (Nash et al., 2002). However, the importance of receptor-G protein coupling efficiency in group I mGlu receptor-mediated Ca signaling has not yet been investigated. It is clear that regulation and localization of Gq/11 proteins could be a key, influencing factor in shaping the Ca signals produced. Studies examining the role of Gq/11 proteins generally, and in conjunction with mGlu receptor signaling, have been facilitated greatly by the generation of Gq/11 knockout mice (for review, see Offermanns, 2003). However, gene deletion studies are limited by the mortality of Gq /G11 doubleknockout mice and also by the possibility that the phenotype of the cells studied may adapt to compensate for the loss of a particular G , as observed for the deletion of other G protein subtypes, including Go (Greif et al., 2000) and G15 (Davignon et al., 2000). Other investigators have successfully used antisense methods to reduce Gq/11 expression; however, these studies often rely on microinjection, making an accurate determination of endogenous Gq and G11 protein expression difficult to ascertain (Macrez-Lepretre et al., 1997; Haley et al., 1998). Determining the relative Gq and G11 expression levels after knockdown is clearly desirable in this type of study. Distinct roles of Gq and G11 in mGlu1a receptor-mediated Ca signaling in Purkinje neurons have recently been shown to result from differential expression levels of these two isoforms (Hartmann et al., 2004). Therefore, it was shown that Gq was solely required for mGlu receptor-dependent synaptic transmission, whereas both Gq and G11 contributed to long-term depression in Purkinje neurons. In the current study, we have used Gq/11 -RNAi and Gq overexpression in combination with single-cell IP3 and Ca 2 imaging as a novel approach to investigate the role of Gq/11 expression in GPCR-mediated signaling. RNAi-knockdown of Gq/11 protein expression was initially characterized in HEK cells stably expressing recombinant M3 mACh receptor. We then demonstrated the effects of RNAi and recombinant Gq expression on IP3 and Ca 2 signals generated by mGlu1 and mGlu5 receptors expressed recombinantly in CHO cells. By altering Gq/11 expression levels, the agonist concentrationdependencies of these GqPCRs were changed. Furthermore, the temporal profiles of Ca signals generated indicate a central role for Gq/11 in defining the nature of the response observed. Materials and Methods Cell Culture and Plasmid Transfection. CHO cells stably expressing the human mGlu1a or mGlu5a receptor under the control of the inducible LacSwitch-II system (Stratagene, La Jolla, CA) were maintained as described previously (Hermans et al., 1998; Nash et al., 2002) and are denoted as CHO-lac-mGlu1 or CHO-lac-mGlu5. HEK cells stably expressing the M3 mACh receptor (HEK-m3) were created and maintained as described previously (Tovey and Willars, 2004). Plasmid containing the fusion construct between eGFP and the pleckstrin homology domain of PLC 1 (eGFP-PHPLC ) was kindly donated by T. Meyer (Stanford University, Stanford, CA). For single cell imaging experiments, CHO-lac-mGlu1/5 or HEK-m3 cells were grown on 25-mm coverslips and cotransfected 72 h before experimentation with 1.8 g of Gq/11 -RNAi, control RNAi, or full-length human Gq and 0.2 g of eGFP-PHPLC (for IP3 imaging) or eGFP (for Ca imaging) using 6 l of GeneJuice (Novagen/EMD Biosciences, Madison, WI) per coverslip. For induction of maximal mGlu receptor expression in CHO cells, the medium was replaced with fresh culture medium containing 100 M IPTG 18 to 20 h before experimentation. For standard SDS-PAGE immunoblotting, HEK-m3 cells were transfected in six-well plates 72 h before experimentation with 2 g of Gq/11 -RNAi or control RNAi using Lipofectamine 2000. CHO cells were transfected in flasks (175 cm) with 10 g of Gq/11 -RNAi or control RNAi using 30 l of GeneJuice and after 24 h, cells were seeded into six-well plates for a further 48 h. For receptor biotinylation, real-time PCR and 6 M urea SDSPAGE analysis, CHO cells were transfected using the Nucleofection system (Amaxa Biosystems, Gaithersburg, MD), according to the manufacturer’s optimized protocol. In brief, 5 10 cells were transfected with 2 g of controlor Gq/11 -RNAi and Program U-23 on the Nucleofector, before seeding cells into six-well plates 72 h before