Increased expression of G11alpha in osteoblastic cells enhances parathyroid hormone activation of phospholipase C and AP-1 regulation of matrix metalloproteinase-13 mRNA

In osteoblasts parathyroid hormone (PTH) stimulates the PTH/PTH-related peptide (PTHrP) receptor (PTH1R) that couples via G(s) to adenylyl cyclase stimulation and via G(11) to phospholipase C (PLC) stimulation. We have investigated the effect of increasing G(11)alpha levels in UMR 106-01 osteoblastic cells by transient transfection with cDNA encoding G(11)alpha on PTH stimulation of PLC and protein kinase C (PKC) as well as PTH regulation of mRNA encoding matrix metalloproteinase-13 (MMP-13). Transfection with G(11)alpha cDNA resulted in a 5-fold increase in PTH-stimulated PLC activity with no change in PTH-stimulated adenylyl cyclase. PTH-induced translocation of PKC-betaI, -delta, and -zeta to the cell membrane and PKC-zeta to the nucleus was also increased. Increased G(11)alpha protein resulted in increased stimulation of MMP-13 mRNA levels at all doses of PTH. There was a 2.5 +/- 0.35 fold increase in maximal PTH-stimulation of c-jun mRNA and smaller but significant increases in c-fos accompanied by increased basal and PTH-stimulated AP-1 binding in cells expressing increased G(11)alpha. Runx-2 mRNA and protein levels were not significantly increased by increased G(11)alpha expression. The increase in PTH stimulation of c-jun, c-fos, and MMP-13 in G(11)alpha-transfected cells were all blocked by bisindolylmaleimide I, a selective inhibitor of PKC. These results demonstrate that regulation of the PLC pathway through the PTH1R is significantly increased by elevating expression of G(11)alpha in osteoblastic cells. This leads to increased PTH stimulation of MMP-13 expression by increased stimulation of AP-1 factors c-jun and c-fos.     

5—HT和NA增强大鼠骶髓后连合核神经元甘氨酸门控Cl^—通道电流由蛋白激...

用制霉菌素穿孔膜片钳方法研究５－ＨＴ和ＮＡ对急性分离的大鼠骶髓后连合核神经元甘氨酸门控氯离子通道电流（ＩＧｌｙ）的调控作用及其胞内机制。发现：（１）５－ＨＴ激活与非胰岛激活蛋白（ＩＡＰ）敏感型Ｇ蛋白偶联的５－ＨＴ２受体亚型,激活磷脂酶Ｃ（ＰＬＣ）,增加甘油二酯（ＤＡＧ）的生成。ＤＡＧ增强不依赖Ｃａ＾２＋的新型ＰＫＣ（ｎＰＫＣ）的活性,从而增强ＩＧｌｙ;（２）ＮＡ激活与ＩＡＰ敏感型Ｇ蛋白偶联的α２受     

Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium.

cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.     

The luteinizing hormone receptor activates phospholipase C via preferential coupling to Gi2.

Binding of lutropin/choriogonadotropin (LH/CG) to its cognate receptor results in the activation of adenylyl cyclase and phospholipase C. This divergent signaling of the LH receptor is based on the independent activation of distinct G protein subfamilies, i.e. , Gs, Gi, and potentially also Gq. To examine the selectivity of LH receptor coupling to phospholipase C beta-activating G proteins, we used an in vivo reconstitution system based on the coexpression of the LH receptor and different G proteins in baculovirus-infected insect cells. In this paper, we describe a refined expression strategy for the LH receptor in insect cells. The receptor protein was inserted into the cell membrane at an expression level of 0.8 pmol/mg of membrane protein. Sf9 cells expressing the LH receptor responded to hCG challenge with a concentration-dependent accumulation of intracellular cAMP (EC50 = 630 nM) but not of inositol phosphates, whereas stimulation of the histamine H1 receptor in Sf9 cells led to increased phospholipase C (PLC) activity. Immunoblotting experiments using G protein-specific antisera revealed the absence of quantitative amounts of alpha i in Sf9 cells, whereas alpha s and alpha q/11 were detected. We therefore attempted to restore the hCG-dependent PLC activation by infection of Sf9 cells with viruses encoding the LH receptor and different G protein alpha subunits. HCG stimulation of cells coexpressing the LH receptor and exogenous alpha i2 resulted in stimulation of PLC activity. In cells coinfected with an alpha i3-baculovirus, hCG challenge led to a minor activation of PLC, whereas no hCG-dependent PLC stimulation was observed in cells coexpressing alpha i1. Most notably, coinfection with baculoviruses encoding alpha q or alpha 11 did not reproduce the PLC activation by the LH receptor. Thus, the murine LH receptor activates adenylyl cyclase via Gs and PLC via selective coupling to Gi2.     

Cardiac specific overexpression of transglutaminase II (G(h)) results in a unique hypertrophy phenotype independent of phospholipase C activation.

Tissue type transglutaminase (TGII, also known as G(h)) has been considered a multifunctional protein, with both transglutaminase and GTPase activity. The role of the latter function, which is proposed as a coupling mechanism between alpha(1)-adrenergic receptors and phospholipase C (PLC), is not well defined. TGII was overexpressed in transgenic mice in a cardiac specific manner to delineated relevant signaling pathways and their consequences in the heart. Cardiac transglutaminase activity in the highest expressing line was approximately 37-fold greater than in nontransgenic lines. However, in vivo signaling to PLC, as assessed by inositol phosphate turnover in [(3)H]myoinositol organ bath atrial preparations, was not increased in the TGII mice at base line or in response to alpha(1)-adrenergic receptor stimulation; nor was protein kinase Calpha (PKCalpha) or PKCepsilon activity enhanced in the TGII transgenic mice. This is in contrast to mice moderately (approximately 5-fold) overexpressing G(alphaq), where inositol phosphate turnover and PKC activity were found to be clearly enhanced. TGII overexpression resulted in a remodeling of the heart with mild hypertrophy, elevated expression of beta-myosin heavy chain and alpha-skeletal actin genes, and diffuse interstitial fibrosis. Resting ventricular function was depressed, but responsiveness to beta-agonist was not impaired. This set of pathophysiologic findings is distinct from that evoked by overexpression of G(alphaq). We conclude that TGII acts in the heart primarily as a transglutaminase, and modulation of this function results in unique pathologic sequelae. Evidence for TGII acting as a G-protein-like transducer of receptor signaling to PLC in the heart is not supported by these studies.     

Role of specific protein kinase C isoforms in modulation of beta1- and beta2-adrenergic receptors

The function of beta-adrenergic receptor (betaAR) is modulated by the activity status of alpha1-adrenergic receptors (alpha1ARs) via molecular crosstalk, and this becomes evident when measuring cardiac contractile responses to adrenergic stimulation. The molecular mechanism underlying this crosstalk is unknown. We have previously demonstrated that overexpression of alpha1B-adrenergic receptor (alpha1BAR) in transgenic mice leads to a marked desensitization of betaAR-mediated adenylyl cyclase stimulation which is correlated with increased levels of activated protein kinase C (PKC) beta, delta and [J. Mol. Cell. Cardiol. 30 (1998) 1827]. Therefore, we wished to determine which PKC isoforms play a role in heterologous betaAR desensitization and also which isoforms of the betaAR were the molecular target(s) for PKC. In experiments using constitutively activated PKC expression constructs transfected into HEK 293 cells also expressing the beta2AR, constitutively active (CA)-PKC overexpression was first confirmed by immunoblots using specific anti-PKC antibodies. We then demonstrated that the different PKC subtypes lead to a decreased maximal cAMP accumulation following isoproterenol stimulation with a rank order of PKCalpha > or = PKCzeta>PKC>PKCbetaII. However, a much more dramatic desensitization of adenylyl cyclase stimulation was observed in cells co-transfected with different PKC isoforms and beta1AR. Further, the modulation of beta1AR by PKC isoforms had a different rank order than for the beta2AR: PKCbetaII>PKCalpha>PKC>PKCzeta. PKC-mediated desensitization was reduced by mutating consensus cAMP-dependent protein kinase (PKA)/PKC sites in the third intracellular loop and/or the carboxy-terminal tail of either receptor. Our results demonstrate therefore that the beta1AR is the most likely molecular target for PKC-mediated heterologous desensitization in the mammalian heart and that modulation of adrenergic receptor activity in any given cell type will depend on the complement of PKC isoforms present.     

OX1 orexin receptors couple to adenylyl cyclase regulation via multiple mechanisms

In this study, the mechanism of OX(1) orexin receptors to regulate adenylyl cyclase activity when recombinantly expressed in Chinese hamster ovary cells was investigated. In intact cells, stimulation with orexin-A led to two responses, a weak (21%), high potency (EC(50) approximately 1 nm) inhibition and a strong (4-fold), low potency (EC(50) = approximately 300 nm) stimulation. The inhibition was reversed by pertussis toxin, suggesting the involvement of G(i/o) proteins. Orexin-B was, surprisingly, almost equally as potent as orexin-A in elevating cAMP (pEC(50) = approximately 500 nm). cAMP elevation was not caused by Ca(2+) elevation or by Gbetagamma. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKCdelta, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of G(s) activity, orexins still elevated cAMP; however, the potencies were greatly increased (EC(50) of orexin-A = approximately 10 nm and EC(50) of orexin-B = approximately 100 nm), and the response was fully dependent on PKCdelta. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-Galpha(s) antibodies. We conclude that OX(1) receptors stimulate adenylyl cyclase via a low potency G(s) coupling and a high potency phospholipase C --> PKC coupling. The former or some exogenous G activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX(1) receptors couple to G(s) almost as efficiently as the orexin-A-activated receptors, in contrast to Ca(2+) elevation and phospholipase C activation, for which orexin-A is 10-fold more potent.     

雌激素的心脏保护作用——与β1-肾上腺素受体的相互作用及其信号通路

雌激素是女性体内主要的类固醇性激素.对于心肌缺血性伤害,切除卵巢的成年雌性大鼠在β-肾上腺素受体激动时,比正常雌性大鼠呈现更严重的心肌损伤;而去卵巢后的雌激素替补组大鼠对β-肾上腺素受体激动时心肌缺血性伤害的反应则又回复到正常雌性大鼠水平,这为雌激素对抗缺血性伤害的心脏保护作用提供了证据.雌激素的这种保护作用是通过下调β1-肾上腺素受体的表达来实现的.也有研究证明,雌激素能抑制蛋白激酶A(protein kinase A,PKA)的表达和活性,PKA是Gs蛋白/腺苷酸环化酶(adenylyl cyclase,AC)/cAMP/PKA通路的第二信使,而该通路最终影响心肌的收缩功能.有初步证据表明雌激素还能抑制β1-肾上腺素受体通路下游的另一种第二信使钙调蛋白激酶Ⅱ-δc(Ca2+/calmodulin kinase Ⅱ-δc,CaMKⅡ-δc)的活性,而CaMKⅡ-δc参与PKA非依赖性的细胞凋亡.即时给予生理浓度雌激素可不通过雌激素受体而直接抑制心肌β1-肾上腺素受体并减弱Ca2+内流.此外,脑研究也显示雌激素能抑制负责调节动脉血压脑区的β广肾上腺素受体活性.因此,雌激素和β1-肾上腺素受体之间的相互作用及其信号通路十分复杂.雌激素不仅主导性别决定,在机体其它功能例如心脏保护方面也具有重要作用.     

Inhibition of phospholipase A2-mediated arachidonic acid release by cyclic AMP defines a negative feedback loop for P2Y receptor activation in Madin-Darby canine kidney D1 cells

In Madin-Darby canine kidney D1 cells extracellular nucleotides activate P2Y receptors that couple to several signal transduction pathways, including stimulation of multiple phospholipases and adenylyl cyclase. For one class of P2Y receptors, P2Y2 receptors, this stimulation of adenylyl cyclase and increase in cAMP occurs via the conversion of phospholipase A2 (PLA2)-generated arachidonic acid (AA) to prostaglandins (e.g. PGE2). These prostaglandins then stimulate adenylyl cyclase activity, presumably via activation of prostanoid receptors. In the current study we show that agents that increase cellular cAMP levels (including PGE2, forskolin, and the beta-adrenergic agonist isoproterenol) can inhibit P2Y receptor-promoted AA release. The protein kinase A (PKA) inhibitor H89 blocks this effect, suggesting that this feedback inhibition occurs via activation of PKA. Studies with PGE2 indicate that inhibition of AA release is attributable to inhibition of mitogen-activated protein kinase activity and in turn of P2Y receptor stimulated PLA2 activity. Although cAMP/PKA-mediated inhibition occurs for P2Y receptor-promoted AA release, we did not find such inhibition for epinephrine (alpha1-adrenergic) or bradykinin-mediated AA release. Taken together, these results indicate that negative feedback regulation via cAMP/PKA-mediated inhibition of mitogen-activated protein kinase occurs for some, but not all, classes of receptors that promote PLA2 activation and AA release. We speculate that receptor-selective feedback inhibition occurs because PLA2 activation by different receptors in Madin-Darby canine kidney D1 cells involves the utilization of different signaling components that are differentially sensitive to increases in cAMP or, alternatively, because of compartmentation of signaling components.     

雌激素的心脏保护作用——与β1-肾上腺素受体的相互作用及其信号通路

雌激素是女性体内主要的类固醇性激素。对于心肌缺血性伤害,切除卵巢的成年雌性大鼠在β-肾上腺素受体激动时,比正常雌性大鼠呈现更严重的心肌损伤：而去卵巢后的雌激素替补组大鼠对β-肾上腺素受体激动时心肌缺血性伤害的反应则又回复到正常雌性大鼠水平,这为雌激素对抗缺血性伤害的心脏保护作用提供了证据。雌激素的这种保护作用是通过下调β1-肾上腺素受体的表达来实现的。也有研究证明,雌激素能抑制蛋白激酶A（protein kinaseA,PKA）的表达和活性,PKA是Gs蛋白／腺苷酸环化酶（adenylyl cyclase,AC）／cAMP／PKA通路的第二信使,而该通路最终影响心肌的收缩功能。有初步证据表明雌激素还能抑制β1-肾上腺素受体通路下游的另一种第二信使钙调蛋白激酶Ⅱ．δc（Ca^2＋/calmodulin kinaseⅡ-δc,CaMKⅡ-δc）的活性,而CaMKII-δc参与PKA非依赖性的细胞凋亡。即时给予生理浓度雌激素可不通过雌激素受体而直接抑制心肌β1-肾上腺素受体并减弱Ca^2＋内流。此外,脑研究也显示雌激素能抑制负责调节动脉血压脑区的β1-肾上腺素受体活性。因此,雌激素和β1-肾上腺素受体之间的相互作用及其信号通路十分复杂。雌激素不仅主导性别决定,在机体其它功能例如心脏保护方面也具有重要作用。     

Protein kinase A-mediated phosphorylation of serine 357 of the mouse prostacyclin receptor regulates its coupling to G(s)-, to G(i)-, and to G(q)-coupled effector signaling.

The prostacyclin receptor (IP) is primarily coupled to G alpha(s)-dependent activation of adenylyl cyclase; however, a number of studies indicate that the IP may couple to other secondary effector systems perhaps in a species-specific manner. In the current study, we investigated the specificity of G protein:effector coupling by the mouse (m) IP overexpressed in human embryonic kidney 293 cells and endogenously expressed in murine erythroleukemia cells. The mIP exhibited efficient G alpha(s) coupling and concentration-dependent increases in cAMP generation in response to the IP agonist cicaprost; however, mIP also coupled to G alpha(i) decreasing the levels of cAMP in forskolin-treated cells. mIP coupling to G alpha(i) was pertussis toxin-sensitive and was dependent on protein kinase (PK) A activation status. In addition, the mIP coupled to phospholipase C (PLC) activation in a pertussis toxin-insensitive, G alpha(i)-, G beta gamma-, and PKC-independent but in a G alpha(q)- and PKA-dependent manner. Whole cell phosphorylation assays demonstrated that the mIP undergoes cicaprost-induced PKA phosphorylation. mIP(S357A), a site-directed mutant of mIP, efficiently coupled to G alpha(s) but failed to couple to G alpha(i) or to efficiently couple to G alpha(q):PLC. Moreover, mIP(S357A) did not undergo cicaprost-induced phosphorylation confirming that Ser(357) is the target residue for PKA-dependent phosphorylation. Finally, co-precipitation experiments permitted the detection of G alpha(s), G alpha(i), and G alpha(q) in the immunoprecipitates of mIP, whereas only G alpha(s) was co-precipitated with mIP(S357A) indicating that Ser(357) of mIP is essential for G alpha(i) and G alpha(q) interaction. Moreover, inhibition of PKA blocked co-precipitation of mIP with G alpha(i) or G alpha(q). Taken together our data indicate that the mIP, in addition to coupling to G alpha(s), couples to G alpha(i) and G alpha(q); however, G alpha(i) and G alpha(q) coupling is dependent on initial cicaprost-induced mIP:G alpha(s) coupling and phosphorylation of mIP by cAMP-dependent PKA where Ser(357) was identified as the target residue for PKA phosphorylation.     

Protein kinase A-induced negative regulation of the corticotropin-releasing hormone R1alpha receptor-extracellularly regulated kinase signal transduction pathway: the critical role of Ser301 for signaling switch and selectivity

Activation of CRH receptors type 1 (CRH-R1) by CRH or urocortin (UCN) leads to stimulation of multiple G proteins with consequent effects on diverse signaling cascades in a tissue-specific manner. In human myometrium and human embryonic kidney (HEK)293 cells, binding of UCN to CRH-R1alpha receptors activates both the Gs and Gq, leading to activation of the adenylyl cyclase/protein kinase A (PKA) and the phospholipase C/protein kinase C and ERK1/2 signaling pathways, respectively. The overall result of these signals is often unpredictable, as these two signaling pathways can interact in many cellular systems, with either potentiation or inhibition of ERK1/2 activity. In the present studies we investigated potential signaling interactions after stimulation of CRH-R1alpha receptors in human cultured pregnant myometrial cells or HEK293 cells overexpressing recombinant CRH-R1alpha receptors. We found that the adenylyl cyclase/PKA pathway has the capacity to markedly decrease UCN-induced ERK1/2 activation, and that these effects were due in part to the ability of PKA to phosphorylate the CRH-R1alpha at position Ser(301) in the third intracellular loop. Mutant CRH-R1alpha receptors with substitutions at position Ser(301), which is the only potential PKA phosphorylation site, were resistant to PKA-dependent phosphorylation and showed altered signaling characteristics, which were dependent upon the amino acid substitution at this position.We conclude that Ser(301), which is located in the third intracellular loop of CRH-R1alpha, is critical for efficient coupling of the receptor to G proteins and to second messenger generation. Phosphorylation by PKA prevents maximal coupling of the CRH-R1alpha to Gq-protein, and thereby reduces activation of ERK 1/2.     

Genetic analysis of the Drosophila Gs(alpha) gene.

One of the best understood signal transduction pathways activated by receptors containing seven transmembrane domains involves activation of heterotrimeric G-protein complexes containing Gs(alpha), the subsequent stimulation of adenylyl cyclase, production of cAMP, activation of protein kinase A (PKA), and the phosphorylation of substrates that control a wide variety of cellular responses. Here, we report the identification of "loss-of-function" mutations in the Drosophila Gs(alpha) gene (dgs). Seven mutants have been identified that are either complemented by transgenes representing the wild-type dgs gene or contain nucleotide sequence changes resulting in the production of altered Gs(alpha) protein. Examination of mutant alleles representing loss-of-Gs(alpha) function indicates that the phenotypes generated do not mimic those created by mutational elimination of PKA. These results are consistent with the conclusion reached in previous studies that activation of PKA, at least in these developmental contexts, does not depend on receptor-mediated increases in intracellular cAMP, in contrast to the predictions of models developed primarily on the basis of studies in cultured cells.     

Calcitonin gene-related peptide stimulates intracellular cAMP via a protein kinase C-controlled mechanism in human ocular ciliary epithelial cells.

Calcitonin gene-related peptides I and II (CGRP I and II) were found to stimulate cAMP levels by approximately 4-6 fold in human nonpigmented ciliary epithelial cells with half-maximal effective concentrations of 20 x 10(-10) and 3 x 10(-10) M, respectively. Prior exposure of cells to 6 x 10(-7) M phorbol 12-myristate, 13-acetate for 15 min resulted in a 40-50% inhibition of CGRP II-dependent cAMP stimulation. Phorbol didecanoate and dioctanoylglycerol also effectively inhibited, whereas 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C, had no effect. Staurosporine, a protein kinase C inhibitor, blocked the inhibition of cAMP formation by phorbol esters. cAMP stimulation by forskolin or cholera toxin was not inhibited by phorbol esters, suggesting that neither a Gs protein nor adenylyl cyclase is the site of inhibition by protein kinase C. These data therefore suggest that CGRP receptors are required for inhibition of adenylate cyclase by protein kinase C.     

Activation of platelet-activating factor receptor-coupled G alpha q leads to stimulation of Src and focal adhesion kinase via two separate pathways in human umbilical vein endothelial cells

Platelet-activating factor (PAF), a phospholipid second messenger, has diverse physiological functions, including responses in differentiated endothelial cells to external stimuli. We used human umbilical vein endothelial cells (HUVECs) as a model system. We show that PAF activated pertussis toxin-insensitive G alpha(q) protein upon binding to its seven transmembrane receptor. Elevated cAMP levels were observed via activation of adenylate cyclase, which activated protein kinase A (PKA) and was attenuated by a PAF receptor antagonist, blocking downstream activity. Phosphorylation of Src by PAF required G alpha(q) protein and adenylate cyclase activation; there was an absolute requirement of PKA for PAF-induced Src phosphorylation. Immediate (1 min) PAF-induced STAT-3 phosphorylation required the activation of G alpha(q) protein, adenylate cyclase, and PKA, and was independent of these intermediates at delayed (30 min) and prolonged (60 min) PAF exposure. PAF activated PLC beta 3 through its G alpha(q) protein-coupled receptor, whereas activation of phospholipase C gamma 1 (PLC gamma 1) by PAF was independent of G proteins but required the involvement of Src at prolonged PAF exposure (60 min). We demonstrate for the first time in vascular endothelial cells: (i) the involvement of signaling intermediates in the PAF-PAF receptor system in the induction of TIMP2 and MT1-MMP expression, resulting in the coordinated proteolytic activation of MMP2, and (ii) a receptor-mediated signal transduction cascade for the tyrosine phosphorylation of FAK by PAF. PAF exposure induced binding of p130(Cas), Src, SHC, and paxillin to FAK. Clearly, PAF-mediated signaling in differentiated endothelial cells is critical to endothelial cell functions, including cell migration and proteolytic activation of MMP2.     

Serine 232 of the alpha(2A)-adrenergic receptor is a protein kinase C-sensitive effector coupling switch.

alpha(2)-adrenergic receptors (alpha(2)AR) couple to multiple effectors including adenylyl cyclase and phospholipase C. We hypothesized that signaling selectivity to these effectors is dynamically directed by kinase-sensitive domains within the third intracellular loop of the receptor. Substitution of Ala for Ser232, which is in the N-terminal region of this loop in the alpha(2A)AR, resulted in a receptor that was markedly uncoupled ( approximately 82% impairment) from stimulation of inositol phosphate accumulation while the capacity to inhibit adenylyl cyclase remained relatively intact. In S232A alpha(2A)AR transfected cell membranes, agonist-promoted [(35)S]GTPgammaS binding was reduced by approximately 50%. Coexpression of modified G proteins rendered insensitive to pertussis toxin revealed that the S232A receptor was uncoupled from both G(i) and G(o). S232 is a potential PKC phosphorylation site, and whole cell phosphorylation studies showed that the mutant had depressed phosphorylation compared to wild type (1.3- vs 2.1-fold/basal). Consistent with S232 directing coupling to phospholipase C, PMA exposure resulted in approximately 67% desensitization of agonist-promoted inositol phosphate accumulation without significantly affecting inhibition of adenylyl cyclase. The dominant effect of mutation or phosphorylation at this site on inositol phosphate as compared to cAMP signaling was found to most likely be due to the low efficiency of signal transduction via phospholipase C vs adenylyl cyclase. Taken together, these results indicate that S232 acts as a selective, PKC-sensitive, modulator of effector coupling of the alpha(2A)AR to inositol phosphate stimulation. This represents one mechanism by which cells route stimuli directed to multifunctional receptors to selected effectors so as to attain finely targeted signaling.     

Apoptotic signaling through the beta -adrenergic receptor. A new Gs effector pathway

Stimulation of beta-adrenergic receptor normally results in signaling by the heterotrimeric G protein G(s), leading to the activation of adenylyl cyclase, production of cAMP, and activation of cAMP-dependent protein kinase (PKA). Here we report that cell death of thymocytes can be induced after stimulation of beta-adrenergic receptor, or by addition of exogenous cAMP. Apoptotic cell death in both cases was observed with the appearance of terminal deoxynucleotidyl transferase-mediated UTP end labeling reactivity and the activation of caspase-3 in S49 T cells. Using thymocytes deficient in either Galpha(s) or PKA, we find that engagement of beta-adrenergic receptors initiated a Galpha(s)-dependent, PKA-independent pathway leading to apoptosis. This alternative pathway involves Src family tyrosine kinase Lck. Furthermore, we show that Lck protein kinase activity can be directly stimulated by purified Galpha(s). Our data reveal a new signaling pathway for Galpha(s), distinct from the classical PKA pathway, that accounts for the apoptotic action of beta-adrenergic receptors.     

A dominant negative Galphas mutant that prevents thyroid-stimulating hormone receptor activation of cAMP production and inositol 1,4,5-trisphosphate turnover: competition by different G proteins for activation by a common receptor

A Ser to Asn mutation at position 54 of the alpha subunit of G(s) (designated N54-alpha(s)) was characterized after transient expression of it with various components of the receptor-adenylyl cyclase pathway in COS-1, COS-7, and HEK 293 cells. Previous studies of the N54-alpha(s) mutant revealed that it has a conditional dominant negative phenotype that prevents hormone-stimulated increases in cAMP without interfering with the regulation of basal cAMP levels (Cleator, J. H., Mehta, N. D., Kurtz, D. K., Hildebrandt, J. D. (1999) FEBS Lett. 243, 205-208). Experiments reported here were conducted to localize the mechanism of the dominant negative effect of the mutant. Competition studies conducted with activated alpha(s)* (Q212L) showed that the N54 mutant did not work down-stream by blocking the interaction of endogenous alpha(s) with adenylyl cyclase. The co-expression of wild type or N54-alpha(s) along with the thyroid-stimulating hormone (TSH) receptor and adenylyl cyclase isotypes differing with respect to betagamma stimulation (AC II or AC III) revealed that the phenotype of the mutant is not dependent upon the presence of adenylyl cyclase isoforms regulated by betagamma. These studies ruled out a downstream site of action of the mutant. To investigate an upstream site of action, N54-alpha(s) was co-expressed with either the TSH receptor that activates both alpha(s) and alpha(q) or with the alpha(1B)-adrenergic receptor that activates only alpha(q). N54-alpha(s) failed to inhibit alpha(1B)-adrenergic receptor stimulation of inositol 1,4,5-trisphosphate production but did inhibit TSH stimulation of inositol 1,4,5-trisphosphate. These results show that G(s) and G(q) compete for activation by the TSH receptor. They also indicate that the N54 protein has a dominant negative phenotype by blocking upstream receptor interactions with normal G proteins. This phenotype is different from that seen in analogous mutants of other G protein alpha subunits and suggests that either regulation or protein-protein interactions differ among G protein alpha subunits.     

G alpha 3 regulates the cAMP signaling system in Dictyostelium.

The Dictyostelium discoideum developmental program is initiated by starvation and its progress depends on G-protein-regulated transmembrane signaling. Disruption of the Dictyostelium G-protein alpha-subunit G alpha 3 (g alpha 3-) blocks development unless the mutant is starved in the presence of artificial cAMP pulses. The function of G alpha 3 was investigated by examining the expression of several components of the cAMP transmembrane signaling system in the g alpha 3- mutant. cAMP receptor 1 protein, cyclic nucleotide phosphodiesterase, phosphodiesterase inhibitor, and aggregation-stage adenylyl cyclase mRNA expression were absent or greatly reduced when cells were starved without exogenously applied pulses of cAMP. However, cAMP receptor 1 protein and aggregation-stage adenylyl cyclase mRNA expression were restored by starving the g alpha 3- cells in the presence of exogenous cAMP pulses. Adenylyl cyclase activity was also reduced in g alpha 3- cells starved without exogenous cAMP pulses compared with similarly treated wild-type cells but was elevated to a level twofold greater than wild-type cells in g alpha 3- cells starved in the presence of exogenous cAMP pulses. These results suggest that G alpha 3 is essential in early development because it controls the expression of components of the transmembrane signaling system.     

Ribozyme approach identifies a functional association between the G protein beta1gamma7 subunits in the beta-adrenergic receptor signaling pathway.

The complex role that the heterotrimeric G proteins play in signaling pathways has become increasingly apparent with the cloning of countless numbers of receptors, G proteins, and effectors. However, in most cases, the specific combinations of alpha and betagamma subunits comprising the G proteins that participate in the most common signaling pathways, such as beta-adrenergic regulation of adenylyl cyclase activity, are not known. The extent of this problem is evident in the fact that the identities of the betagamma subunits that combine with the alpha subunit of Gs are only now being elucidated almost 20 years after its initial purification. In a previous study, we described the first use of a ribozyme strategy to suppress specifically the expression of the gamma7 subunit of the G proteins, thereby identifying a specific role of this protein in coupling the beta-adrenergic receptor to stimulation of adenylyl cyclase activity in HEK 293 cells. In the present study, we explored the potential utility of a ribozyme approach directed against the gamma7 subunit to identify functional associations with a particular beta and alphas subunit of the G protein in this signaling pathway. Accordingly, HEK 293 cells were transfected with a ribozyme directed against the gamma7 subunit, and the effects of this manipulation on levels of the beta and alphas subunits were determined by immunoblot analysis. Among the five beta alphas subunits detected in these cells, only the beta1 subunit was coordinately reduced following treatment with the ribozyme directed against the gamma7 subunit, thereby demonstrating a functional association between the beta1 and gamma7 subunits. The mechanism for coordinate suppression of the beta1 subunit was due to a striking change in the half-life of the beta1 monomer versus the beta1 heterodimer complexed with the gamma7 subunit. Neither the 52- nor 45-kDa subunits were suppressed following treatment with the ribozyme directed against the gamma7 subunit, thereby providing insights into the assembly of the Gs heterotrimer. Taken together, these data show the utility of a ribozyme approach to identify the role of not only the gamma subunits but also the beta subunits of the G proteins in signaling pathways.