RGS14, a GTPase-Activating Protein for Gia, Attenuates Gia- and G13a-Mediated Signaling Pathways

Regulator of G protein signaling (RGS) proteins are a family of approximately 20 proteins that negatively regulate signaling through heterotrimeric G protein-coupled receptors. The RGS proteins act as GTPase-activating proteins (GAPs) for certain Ga subunits and as effector antagonists for Gqa. Mouse RGS14 encodes a 547-amino-acid protein with an N-terminal RGS domain, which is highly expressed in lymphoid tissues. In this study, we demonstrate that RGS14 is a GAP for Gia subfamily members and it attenuates interleukin-8 receptor-mediated mitogen-activated protein kinase activation. However, RGS14 does not exhibit GAP activity toward Gsa or Gqa nor does it regulate Gsaor Gqa-mediated signaling pathways. Although RGS14 does not act as a GAP for G12/13a, it impairs c-fos serum response element activation induced by either a constitutively active mutant of G13a (G13aQ226L) or by carbachol stimulation of muscarinic type 1 receptors. An RGS14 mutant (EN92/93AA), which does not block Gia-linked signaling, also inhibits serum response element activation. RGS14 localizes predominantly in the cytosol, but it can be recruited to membranes by expression of G13aQ226L. Although RGS14 is constitutively expressed in lymphoid cells, agents that activate B or T lymphocytes further enhance its levels. Taken together, our results suggest that signals generated after lymphocyte activation may via RGS14 directly impinge on Giaor G13amediated cellular processes in lymphocytes, such as adhesion and migration. Extracellular signals such as hormones, neurotransmitters, and chemokines that stimulate heptahelical receptor are transmitted via heterotrimeric G proteins, signal transducers, resulting in regulation of a variety of enzymes and ion channels (Hamm and Gilchrist, 1996). One way to control the duration and sensitivity of the G protein-mediated signaling is to alter the intrinsic GTPase activity of Ga subunits. Regulator of G protein signaling (RGS) proteins are a newly described family of approximately 20 proteins that can act as GTPase-activating proteins (GAPs) for certain Ga subunits, thereby negatively regulating signaling through G protein-coupled receptors (GPCR). They were originally identified as functional homologs of yeast Sst2p and EGL10 of Caenorhabditis elegans, and subsequently shown to impair signaling mediated via GPCRs in mammalian systems (Druey et al., 1996, for reviews, see Berman and Gilman, 1998; Kehrl, 1998). RGS proteins have a highly conserved, 120-amino-acid core region called “RGS domain”. Solution of a cocrystal structure of RGS4 and Gia1 revealed that critical residues in the RGS domain stabilize the flexible switch regions of Ga proteins in the transition state of GTP hydrolysis, thus lowering the activation energy barrier (Tesmer et al., 1997). The RGS domain contains all of the crucial elements necessary for the GAP activity. Furthermore, alteration of critical residues in RGS4 located at the contact sites between RGS4 and Gia1 completely abolished its GAP activity and ability to bind to Gia (Druey and Kehrl, 1997; Srinivasa et al., 1998). Although it seems redundant that 20 or so RGS proteins should all act as GAPs for Gia and Gqa, clear differences among the family members are emerging. RGS proteins differ in their molecular masses (;20 to 150 kDa), their specificities for various Ga subfamily members, their tissueor cell-specific expression patterns, their subcellular localization, and their types of post-translational modifications (Zerangue and Jan, 1998; Druey et al., 1998). Furthermore, a variety of proteins that interact with specific RGS family members has been identified. For example, RAP1/2, GIPC, Rho, and Gb5 interact with RGS14, GAIP, p115 RhoGEF, and RGS7, respectively (Cabrera et al., 1998; De Vries et al., 1998; Hart et al., 1998; Traver et al., 2000). Finally, RGSr (RGS16) is induced by the tumor suppressor protein p53, ABBREVIATIONS: RGS, regulator of G protein signaling; GAP, GTPase-activating protein; GPCR, G protein-coupled receptor; FCS, fetal calf serum; IL, interleukin; HA, hemagglutinin; ERK, extracellular signal-related kinase; MAP, mitogen-activated protein; MBP, myelin basic protein; PAGE, polyacrylamide gel electrophoresis; M1, muscarinic type 1; PLC, phospholipase C; CREB, cAMP response element-binding protein; TCR, T cell receptor; BCR, B cell receptor; PAF, platelet-activating factor; SRE, serum response element. 0026-895X/00/030569-00 MOLECULAR PHARMACOLOGY Vol. 58, No. 3 U.S. Government work not protected by U.S. copyright 85/848106 Mol Pharmacol 58:569–576, 2000 Printed in U.S.A. 569 at A PE T Jornals on O cber 9, 2017 m oharm .aspeurnals.org D ow nladed from suggesting an involvement in its role in regulating apoptosis or cell cycle arrest (Buckbinder et al., 1997). There are four salient questions in studying the RGS proteins: 1) What specificities do RGS proteins exhibit for various G proteins? 2) What other signaling molecules do RGS proteins interact with? What is the significance of that interaction? 3) How are the RGS proteins regulated? and 4) What are the in vivo roles of different RGS proteins? In this report, we characterized the RGS14 protein to address the above-mentioned questions. RGS14 was originally identified as RAP1/2-interacting protein in yeast 2-hybrid screen (Traver et al., 2000) and by degenerate polymerase chain reaction cloning (Snow et al., 1997). We find that the GAP activity of RGS14 is directed at members of Gia subfamily, although RGS14 inhibits both Giaand G13a-linked signaling pathways. To understand the physiological function(s) of RGS14 protein, we studied tissueand cell-specific expression patterns, and subcellular localization of RGS14. In addition, because of the expression of RGS14 in lymphocytes, we studied the effects on RGS14 expression of signals that trigger either B or T cells. Materials and Methods Cell Culture, Transfection, and Lymphocyte Purification. All lymphoid cells were maintained in RPMI 1640 (Life Technologies Inc., Gaithersburg MD) supplemented with 10% fetal calf serum (FCS). Human embryonic kidney 293T and monkey kidney COS-7 cells were grown in Dulbecco’s modified Eagle’s medium containing 10% FCS. Transfection of the 293T and COS-7 cells was performed by using calcium-phosphate precipitation method or by using Lipofectamine (Life Technologies Inc.). The total amount of plasmid DNA for each transfection was always normalized with vector DNA. Peripheral leukocytes were isolated from blood of healthy human donors by ficoll hypaque (Pharmacia, Uppsala, Sweden) density centrifugation. T cells were separated by adsorption to sheep red blood cells. B cells were purified from the remaining cells by the removal of CD14-positive cells with a CD14 mouse monoclonal antibody (Pharmingen, San Diego, CA) and goat anti-mouse dynabeads (Dynal, Oslo, Norway). The purity of the T and B fractions was verified by a fluorescence-activated cell sorter Calibur flow cytometer after staining with monoclonal antibodies directed against CD3 and CD19 (Pharmingen). Purified T cells were stimulated with CD3 (0.1 mg/ml; Pharmingen) and interleukin (IL)-2 (20%; Hemagen Diagnostics, Inc., Waltham, MA) every 3 days to maintain cell viability and purified B cells stimulated with anti-IgM F(ab9)2 fragment (20 mg/ml; ICN Pharmaceuticals, Inc., Costa Mesa, CA) in conjunction with CD40 (1 mg/ml; Pharmingen). Forskolin and ionomycin were purchased from Sigma (St. Louis, MO) Production of Recombinant RGS14 Protein. We generated hexa-histidine-tagged RGS14 protein by subcloning a cDNA fragment that would encode either full-length RGS14 or the RGS14 RGS domain (W64 to E187) into NdeI and BamHI restriction sites of pET15b vector (Novagen, Inc., Madison, WI). The resulting constructs were used to overexpress RGS14 proteins in an Escherichia coli strain BL21 (DE3) by induction with 1 mM isopropyl b-D-thiogalactoside for 1 h. Histidine-tagged RGS14 recombinant proteins were purified with nickel-nitrilotriacetic acid resin (Qiagen, Chatsworth CA) as described in manufacturer’s protocol (Novagen, Inc.). RGS14 Antiserum, Immunoblotting, and Immunofluorescence. Full-length mouse recombinant RGS14 was used to generate anti-RGS14 antiserum in rabbit and immunoblotting (1:1000 dilution) was performed as previously described (Druey et al., 1998). For immunofluorescent cytochemistry, 293 cells were transfected with hemagglutinin (HA)-epitope tagged RGS14 (0.5 mg) and grown in culture dishes containing glass coverslips overnight. Cells were washed in PBS once and then fixed in 50% methanol/50% acetone for 1 h at 4°C. The cover slips were washed twice with PBS and incubated in 10% goat serum plus 2% BSA in PBS for 1 h. Each coverslip was then placed in 2% BSA in PBS containing anti-RGS14 antiserum (1:800 dilution) for 2 h at room temperature. The coverslips were washed, incubated with Cy3-conjugated goat anti-rabbit immunoglobulins (Jackson ImmunoResearch Laboratory, Inc., West Grove, PA) for 1 h. The coverslips were washed again with PBS, mounted on silanized glass slides, and examined with a fluorescence