The G protein coupled to the thromboxane A2 receptor in human platelets is a member of the novel Gq family.

The thromboxane A2 (TXA2) receptor in human platelets is coupled to a pertussis toxin-insensitive G protein whose identity has remained unknown. Candidates for this role include the atypical G protein known as Gz and members of a recently discovered G protein family known as Gq. Because of the proven utility of antibodies directed against the C terminus of G protein alpha subunits as functional probes, we prepared an antibody against a synthetic decapeptide corresponding to the C-terminal sequence shared by alpha 11 and alpha q, two members of the new family. This antibody (QL) does not recognize known alpha subunits but selectively binds to a 42-kDa protein in a variety of tissues, including human platelet membranes. QL and two other C-terminal antibodies, QN and AS, known to recognize alpha z and alpha i2, respectively, were tested for their ability to block agonist-stimulated GTPase activity in human platelet membranes. Pretreatment of platelet membranes with AS has previously been shown to interfere with alpha 2 adrenergic receptor-mediated inhibition of adenylylcyclase. As expected, only AS antibody produced inhibition of alpha 2 receptor-stimulated GTPase. Pretreatment of membranes with QL, but not QN or AS, caused marked inhibition of TXA2 receptor-stimulated GTPase. This identifies the G protein coupled to human platelet TXA2 receptors as a member of the novel Gq family.     

Interaction of early growth response protein 1 (Egr-1), specificity protein 1 (Sp1), and cyclic adenosine 3'5'-monophosphate response element binding protein (CREB) at a proximal response element is critical for gastrin-dependent activation of the chromogranin A promoter

Recently, binding of specific protein 1 (Sp1) and cAMP response element binding protein (CREB) to a GC-rich element at -92/-62 has been identified as a critical step in gastrin-dependent regulation of the chromogranin A (CgA) gene in gastric epithelial cells. Here we demonstrate that binding of early growth response protein 1 (Egr-1) to the distal part of the -92/-62 site is also required for gastrin-dependent CgA transactivation. Gastrin elevated cellular and nuclear Egr-1 levels in a time-dependent manner and also increased Egr-1 binding to the CgA -92/-73 region. Disruption of this site reduced gastrin responsiveness without influencing basal promoter activity, while loss of Sp1 and/or CREB binding sites diminished basal and gastrin-stimulated CgA promoter activity. Ectopic Egr-1 overexpression potently stimulated the CgA promoter, whereas coexpression of Egr-1 with Sp1 and/or CREB resulted in additive effects. Functional analysis of Sp1-, Egr-1-, or CREB-specific promoter mutations in transfection studies confirmed the tripartite organization of the CgA -92/-62 element. Signaling studies revealed that MAPK kinase 1 (MEK1)/ERK1/2 cascades are critical for gastrin-dependent Egr-1 protein accumulation as well as Egr-1 binding to the CgA promoter. Our studies for the first time identify Egr-1 as a nuclear target of gastrin and show that functional interplay of Egr-1, Sp1, and CREB is indispensable for gastrin-dependent CgA transactivation in gastric epithelial cells.     

G protein-coupled receptor Kinase 2/G alpha q/11 interaction. A novel surface on a regulator of G protein signaling homology domain for binding G alpha subunits

G protein-coupled receptors (GPCRs) transduce cellular signals from hormones, neurotransmitters, light, and odorants by activating heterotrimeric guanine nucleotide-binding (G) proteins. For many GPCRs, short term regulation is initiated by agonist-dependent phosphorylation by GPCR kinases (GRKs), such as GRK2, resulting in G protein/receptor uncoupling. GRK2 also regulates signaling by binding G alpha(q/ll) and inhibiting G alpha(q) stimulation of the effector phospholipase C beta. The binding site for G alpha(q/ll) resides within the amino-terminal domain of GRK2, which is homologous to the regulator of G protein signaling (RGS) family of proteins. To map the Galpha(q/ll) binding site on GRK2, we carried out site-directed mutagenesis of the RGS homology (RH) domain and identified eight residues, which when mutated, alter binding to G alpha(q/ll). These mutations do not alter the ability of full-length GRK2 to phosphorylate rhodopsin, an activity that also requires the amino-terminal domain. Mutations causing G alpha(q/ll) binding defects impair recruitment to the plasma membrane by activated G alpha(q) and regulation of G alpha(q)-stimulated phospholipase C beta activity when introduced into full-length GRK2. Two different protein interaction sites have previously been identified on RH domains. The G alpha binding sites on RGS4 and RGS9, called the "A" site, is localized to the loops between helices alpha 3 and alpha 4, alpha 5 and alpha 6, and alpha 7 and alpha 8. The adenomatous polyposis coli (APC) binding site of axin involves residues on alpha helices 3, 4, and 5 (the "B" site) of its RH domain. We demonstrate that the G alpha(q/ll) binding site on the GRK2 RH domain is distinct from the "A" and "B" sites and maps primarily to the COOH terminus of its alpha 5 helix. We suggest that this novel protein interaction site on an RH domain be designated the "C" site.     

A role for AKAP (A kinase anchoring protein) scaffolding in the loss of a cyclic adenosine 3',5'-monophosphate inhibitory response in late pregnant rat myometrium

During pregnancy in the rat, there is a change in the ability of chlorophenylthio (CPT)-cAMP to inhibit myometrial phosphatidylinositide turnover. This is accompanied by a change in the association of proteins with a plasma membrane A kinase anchoring protein (AKAP). Both CPT-cAMP and isoproterenol inhibited oxytocin-stimulated phosphatidylinositide turnover on days 12 through 20 of gestation, whereas neither agent had an effect on day 21. Accompanying this change was a dramatic decrease in the concentration and activity of cAMP-dependent protein kinase [protein kinase A (PKA)] and an increase in the concentration of protein phosphatase 2B (PP2B) in plasma membranes from day 21 compared with day 19 pregnant rats. In contrast, both PKA and PP2B concentrations and activities increased in total myometrial homogenates. Both PKA and PP2B coimmunoprecipitated with an antibody against the 150-kDa AKAP found in rat myometrial plasma membranes. More PKA was associated with AKAP150 on day 19 than on day 21, while the reverse was true for PP2B. Disruption of PKA/AKAP association in day 19 pregnant rat myometrial cells with the specific interaction inhibitor peptide S-Ht31 resulted in the loss of the cAMP-inhibitory effect on phosphatidylinositide turnover. PP2B activity in myometrial homogenates dephosphorylated PLCbeta3, a PKA substrate targeted in the inhibition of Galphaq-stimulated phosphatidylinositide turnover. The dramatic loss of the cAMP-inhibitory effect on day 21 of pregnancy may alter the balance between uterine contraction and relaxation near parturition. The changes in the relative concentrations of PKA and PP2B associated with AKAP150 are consistent with a functional role for AKAP150 scaffolding in the alteration of cellular signaling.     

A novel catecholamine-activated adenosine cyclic 3',5'-phosphate independent pathway for beta-adrenergic receptor phosphorylation in wild-type and mutant S49 lymphoma cells: mechanism of homologous desensitization of adenylate cyclase

Virtually all known biological actions stimulated by beta-adrenergic and other adenylate cyclase coupled receptors are mediated by cAMP-dependent protein kinase. Nonetheless, "homologous" or beta-adrenergic agonist-specific desensitization does not require cAMP. Since beta-adrenergic receptor phosphorylation may be involved in desensitization, we studied agonist-promoted receptor phosphorylation during homologous desensitization in wild-type S49 lymphoma cells (WT) and two mutants defective in the cAMP-dependent pathway of beta-agonist-stimulated protein phosphorylation (cyc- cannot generate cAMP in response to beta-adrenergic agonists; kin- lacks cAMP-dependent kinase). All three cell types demonstrate rapid, beta-adrenergic agonist-promoted, stoichiometric phosphorylation of the receptor which is clearly not cAMP mediated. The amino acid residue phosphorylated is solely serine. These data demonstrate, for the first time, that catecholamines can promote phosphorylation of a cellular protein (the beta-adrenergic receptor) via a cAMP-independent pathway. Moreover, the ability of cells with mutations in the adenylate cyclase-cAMP-dependent protein kinase pathway to both homologously desensitize and phosphorylate the beta-adrenergic receptors provides very strong support for the notion that receptor phosphorylation may indeed be central to the molecular mechanism of desensitization.