Species difference in the G protein selectivity of the human and bovine A1-adenosine receptor

The purified bovine brain A1-adenosine receptor has previously been shown to discriminate among closely related G protein alpha-subunits. To obtain analogous information for the human receptor, the cDNA coding for the human A1-adenosine receptor was inserted into a plasmid placing the synthesis of the receptor protein under the control of the MalE promoter. Following induction by maltose, active receptor accumulated in Escherichia coli membranes. Binding of the antagonist 8-[3H]cyclopentyl-1,3-dipropylxanthine to E. coli membranes (KD approximately 2 nM, Bmax approximately 0.2-0.4 pmol/mg) showed the appropriate pharmacological profile. Incubation of E. coli membranes with purified Go,i-reconstituted guanine nucleotide-sensitive high affinity binding of the agonist (-)[125I] N6-3-(iodo-4-hydroxyphenylisopropyl)adenosine to the receptor (KD approximately 1 nM). In the presence of purified beta gamma-subunit, the recombinant receptor interacted equally well with the recombinant G protein alpha-subunits Gi alpha-1, Gi alpha-2, Gi alpha-3; G(o) alpha displayed a lower affinity for the receptor while Gs alpha was inactive. Parallel experiments were carried out in bovine and human brain membranes pretreated with N-ethylmaleimide to inactivate the endogenous G(o)/Gi proteins; Gi alpha-3 was most potent in reconstituting 125I-HPIA binding to bovine membranes, while Gi alpha-1, Gi alpha-2, and G(o) alpha displayed similar affinities. However, in human membranes, Gi alpha-1, Gi alpha-2, and Gi alpha-3, were equipotent and high concentrations of G(o) alpha were required to promote 125I-HPIA binding. These observations show (i) that functional human A1-adenosine receptors were synthesized in E. coli; (ii) that the pattern of G protein coupling is identical for the recombinant human A1-receptor and its counterpart in the native membrane; (iii) and that species differences between bovine and human receptor exist not only in their pharmacological profile but also in their G protein specificity suggesting that species homologues of receptors may use different signaling mechanisms.     

Rescue of functional interactions between the alpha2A-adrenoreceptor and acylation-resistant forms of Gi1alpha by expressing the proteins from chimeric open reading frames

Co-expression of the alpha2A-adrenoreceptor with a pertussis toxin-resistant (C351G), but not with an also palmitoylation-resistant (C3S/C351G), form of the alpha subunit of Gi1 resulted in agonist-induced, pertussis toxin-independent, GTP hydrolysis. Construction and expression of a chimeric fusion protein between the receptor and C351G Gi1alpha generated a membrane protein in which the G protein element was activated by receptor agonist. An equivalent fusion protein containing C3S/C351G Gi1alpha rescued the ability of receptor agonist to activate this mutant. Fusion proteins of a palmitoylation-resistant (C442A) alpha2A-adrenoreceptor and either C351G or C3S/C351G Gi1alpha also responded effectively to agonist. Myristoylation resistant (G2A/C351G) and combined acylation-resistant (G2A/C3S/C351G) mutants of Gi1alpha are cytosolic proteins. Expression of these as chimeric alpha2A-adrenoreceptor-G protein fusions restored membrane localization and activation of the G protein by receptor agonist. These studies demonstrate the general utility of generating chimeric fusion proteins to examine receptor regulation of G protein function and that the lack of functional activation of acylation-negative G proteins by a co-expressed receptor is related to deficiencies in cellular targeting and location rather than an inherent incapacity to produce appropriate protein-protein interactions and signal transmission.     

Signal transduction through trimeric G proteins in megakaryoblastic cell lines

The biogenesis of trimeric G proteins was investigated by measurement of the expression of alpha-subunits in the megakaryoblastic cell lines MEG-01, DAMI, and CHRF-288-11, representing stages of increasing maturation, and compared with platelets. Megakaryoblasts and platelets contained approximately equal amounts of Gi alpha-1/2, Gi alpha-3, Gq alpha, and G12 alpha protein. Maturation was accompanied by (1) downregulation of mRNA for Gs alpha and disappearance of iloprost-induced Ca2+ mobilization, (2) upregulation of the long form of Gs alpha protein (Gs alpha-L) and an increase in iloprost-induced cAMP formation, and (3) upregulation of G16 alpha mRNA and G16 alpha protein and appearance of thromboxane A2-induced signaling (Ca2+ mobilization and stimulation of prostaglandin I2-induced cAMP formation). Gz alpha protein was absent in the megakaryoblasts despite weak expression of Gz alpha mRNA in DAMI and relatively high levels of Gz alpha mRNA and Gz alpha protein in platelets. These findings reveal major changes in G protein-mediated signal transduction during megakaryocytopoiesis and indicate that G16 alpha couples the thromboxane receptor to phospholipase C beta.     

Agonist-mediated downregulation of G alpha i via the alpha 2-adrenergic receptor is targeted by receptor-Gi interaction and is independent of receptor signaling and regulation

One mechanism of long-term agonist-promoted desensitization of alpha2AR function is downregulation of the cellular levels of the alpha subunit of the inhibitory G protein, Gi. In transfected CHO cells expressing the human alpha2AAR, a 40.1 +/- 3.3% downregulation of Galphai2 protein occurred after 24 h of exposure of the cells to epinephrine, which was not accompanied by a decrease in Galphai2 mRNA. The essential step that targets Gi for degradation by agonist occupancy of the receptor was explored using mutated alpha2AAR lacking specific structural or functional elements. These consisted of 5HT1A receptor and beta2AR sequences substituted at residues 113-149 of the second intracellular loop and 218-235 and 355-371 of the N- and C-terminal regions of the third intracellular loop (altered Gi and Gs coupling), deletion of Ser296-299 (absent GRK phosphorylation), and substitution of Cys442 (absent palmitoylation and receptor downregulation). Of these mutants, only those with diminished Gi coupling displayed a loss of agonist-promoted Gi downregulation, thus excluding Gs coupling and receptor downregulation, palmitoylation, and phosphorylation as necessary events. Furthermore, coupling-impaired receptors consisting of mutations in the second or third loops ablated Gi downregulation, suggesting that a discreet structural motif of the receptor is unlikely to represent a key element in the process. While pertussis toxin ablated Gi downregulation, blocking downstream intracellular consequences of alpha2AAR activation or mimicking these pathways by heterologous means failed to implicate cAMP/adenylyl cyclase, phospholipase C, phospholipase D, or MAP kinase pathways in alpha2AAR-mediated Gi downregulation. Taken together, agonist-promoted Gi downregulation requires physical alpha2AAR-Gi interaction which targets Gi for degradation in a manner that is independent of alpha2AAR trafficking, regulation, or second messengers.     

Alpha adrenergic receptor subtypes involved in prostaglandin synthesis are coupled to Ca++ channels through a pertussis toxin-sensitive guanine nucleotide-binding protein

Previously, we have shown that alpha-2C and alpha-1A adrenergic receptors (AR) stimulate prostacyclin (PGI2) synthesis through a pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) in vascular smooth muscle cells (VSMC). The purpose of this study was to assess the role of Ca++ in PGI2 production elicited by alpha-AR activation and to investigate the modulation of the Ca++ channel by G proteins coupled to these alpha-AR in VSMC. PGI2 was measured as immunoreactive 6-keto-PGF1 alpha by radioimmunoassay and cytosolic calcium ([Ca++]i) by spectrofluorometry using fura-2. Norepinephrine, methoxamine and UK-14304 enhanced 6-keto-PGF1 alpha production and [Ca++]i, which was inhibited by depletion of extracellular Ca++ and by Ca++ channel antagonists (verapamil, nifedipine and PN 200-110). Moreover, the Ca++ channel activator Bay K 8644 increased 6-keto-PGF1 alpha production in a nifedipine-sensitive manner, indicating the involvement of dihydropyridine-sensitive Ca++ channels in VSMC. Pertussis toxin inhibited AR agonist-induced 6-keto-PGF1 alpha production and the increase in [Ca++]i. Alpha AR agonists increase Ca++ influx in the presence of guanosine 5'-0-(2- thiodiphosphate) (GTP-gamma-S), and this effect was blocked in the presence of guanine 5'-O-(2-thiodiphosphate) (GDP-beta-S) and antiserum against Gi alpha 1-2 protein in reversibly permeabilized cells with beta-escin. VSMC of rabbit aortae contain a G protein(s) that was recognized by Gi alpha 1-2 but not Gi alpha 3 or G0 antibodies at 1:200 dilution. The calmodulin inhibitor W-7 blocked AR agonist and Bay K 8644-stimulated 6-keto-PGF1 alpha production. The phospholipase A2 inhibitors 7,7-dimethyleicosadienoic acid and oleoyloxyethyl phosphocholine but not phospholipase C inhibitor U-73122 reduced 6-keto-PGF1 alpha production in VSMC. These data suggest that a pertussis toxin-sensitive G protein, probably Gi alpha 1-2, coupled to alpha AR regulates Ca++ influx, which, in turn, by interacting with calmodulin, increases phospholipase A2 activity to release arachidonic acid for PGI2 synthesis in VSMC of rabbit aortae.     

Selective activation of G protein subtypes in the vomeronasal organ upon stimulation with urine-derived compounds

Chemosensory neurons in the vomeronasal organ (VNO) detect pheromones related to social and reproductive behavior in most terrestrial vertebrates. Current evidence indicate that the chemoelectrical transduction process is mediated by G protein-coupled second messenger cascades. In the present study, attempts were made to identify the G protein subtypes which are activated upon stimulation with urinary pheromonal components. G protein-specific antibodies were employed to interfere specifically with inositol 1,3,4-trisphosphate formation induced by urinary stimuli and to immunoprecipitate Galpha-subunits, activation dependently labeled with [alpha-32P]GTP azidoanilide. The results of both experimental approaches indicate that stimulation of female VNO membrane preparations with male urine samples induces activation of Gi as well as Go subtypes. Experiments using different fractions of urine revealed that upon stimulation with lipophilic volatile odorants, only Gi proteins were activated, whereas Go activation was elicited by alpha2u-globulin, a major urinary protein, which is a member of the lipocalin superfamily. Since each G protein subtype is stereotypically coexpressed with one of the two structurally different candidate pheromone receptors (V1R and V2R), the results provide the first experimental evidence that V1Rs coexpressed with Gi may be activated by lipophilic probably volatile odorants, whereas V2Rs coexpressed with Go seem to be specialized to interact with pheromonal components of proteinaceous nature.     

RGS4 inhibits Gq-mediated activation of mitogen-activated protein kinase and phosphoinositide synthesis

Recombinant regulators of G protein-signaling (RGS) proteins stimulate hydrolysis of GTP by alpha subunits of the Gi family but have not been reported to regulate other G protein alpha subunits. Expression of recombinant RGS proteins in cultured cells inhibits Gi-mediated hormonal signals probably by acting as GTPase-activating proteins for Galphai subunits. To ask whether an RGS protein can also regulate cellular responses mediated by G proteins in the Gq/11 family, we compared activation of mitogen-activated protein kinase (MAPK) by a Gq/11-coupled receptor, the bombesin receptor (BR), and a Gi-coupled receptor, the D2 dopamine receptor, transiently co-expressed with or without recombinant RGS4 in COS-7 cells. Pertussis toxin, which uncouples Gi from receptors, blocked MAPK activation by the D2 dopamine receptor but not by the BR. Co-expression of RGS4, however, inhibited activation of MAPK by both receptors causing a rightward shift of the concentration-effect curve for both receptor agonists. RGS4 also inhibited BR-stimulated synthesis of inositol phosphates by an effector target of Gq/11, phospholipase C. Moreover, RGS4 inhibited inositol phosphate synthesis activated by addition of AlF4- to cells overexpressing recombinant alphaq, probably by binding to alphaq.GDP.AlF4-. These results demonstrate that RGS4 can regulate Gq/11-mediated cellular signals by competing for effector binding as well as by acting as a GTPase-activating protein.     

Role of basic fibroblast growth factor in the regulation of rat basilar artery tone in vivo

An antisense oligodeoxynucleotide directed against the 5'-untranslated region of MOR-1 blocks the analgesic actions of the mu 1 analgesics morphine and [D-Ala2,D-Leu5]enkephalin (DADL) when they are microinjected into the periaqueductal gray. In contrast, morphine-6 beta-glucuronide (M6G) analgesia is unaffected by this treatment. Antisense oligodeoxynucleotides directed against distinct Gi alpha subunits also distinguish between morphine and M6G analgesia. A probe targeting Gi alpha 2 blocks morphine analgesia, as previously reported, but is inactive against M6G analgesia. Conversely, an antisense oligodeoxynucleotide against Gi alpha 1 inhibits M6G analgesia without affecting morphine analgesia. The antisense oligodeoxynucleotide directed against G(o)alpha is ineffective against both compounds. These results confirm the prior association of Gi alpha 2 with morphine analgesia and strongly suggests that M6G acts through a different opioid receptor, as revealed by its insensitivity towards the MOR-1 antisense probe and differential sensitivity towards G-protein alpha subunit antisense oligodeoxynucleotides.     

Overexpression of Gi-proteins precedes the development of DOCA-salt-induced hypertension: relationship with adenylyl cyclase

OBJECTIVE: In the present studies, we have investigated if aorta, like heart from deoxycorticosterone acetate (DOCA)-salt hypertensive rats, (HR) also exhibit enhanced expression of G-protein levels and if these alterations occur before or after the development of blood pressure. METHODS: Sprague-Dawley rats treated with DOCA-salt or vehicle for 1, 2, 3 and 4 weeks were used for these studies. The levels of inhibitory guanine nucleotide regulatory proteins (Gi alpha-2, Gi alpha-3) and G beta proteins were determined by immunoblotting, whereas the levels of Gi alpha-2 and Gi alpha-3 and adenylyl cyclase type V enzyme mRNA were determined by Northern-blotting techniques. RESULTS: The blood pressure was significantly increased in DOCA-salt-treated rats as compared to sham-operated rats after 2 to 4 weeks of treatment; whereas no change in blood pressure was observed after 1 week of treatment (prehypertensive state). However, the levels of Gi alpha-2, Gi alpha-3 and G beta proteins and Gi alpha-2 and Gi alpha-3 mRNA were significantly enhanced in hearts and aorta from DOCA-salt treated rats after 1 week of treatment and remained elevated up to 4 weeks of treatment. In addition, the Gi-mediated inhibitions of adenylyl cyclase by Angiotensin II (Ang II) and C-ANF4-23 were also greater in DOCA-salt-treated rats as compared to sham-operated rats after 1 week and longer periods of treatments (2 to 4 weeks). On the other hand, the levels of Gs alpha were not altered up to 2 weeks of DOCA-salt treatment but significantly decreased in rats treated for 3 and 4 weeks. Furthermore, the stimulatory effects of guanine 5'-[gamma-thio]triphosphate (GTP gamma S), isoproterenol and forskolin on adenylyl cyclase were decreased in both hearts and aorta from DOCA-salt-treated rats after 1 to 4 weeks of treatment as compared to sham-operated rats. The mRNA levels of adenylyl cyclase, type V enzyme in hearts from DOCA-salt treated rats were significantly decreased after 3 and 4 weeks of DOCA-salt treatment but not in rats treated for 1 or 2 weeks. CONCLUSIONS: These results indicate that the enhanced expression of Gi alpha-2 and Gi alpha-3 precedes the development of blood pressure in DOCA-salt-induced hypertension. It can thus be suggested that the increased levels of Gi proteins and resulting decreased levels of cAMP may be one of the factors that contribute to the impaired cardiac contractility and increased vascular tone in DOCA-salt hypertension.     

Activation of various subtypes of G-protein alpha subunits by partial agonists of the adenosine A1 receptor

The activation of different G protein subtypes by the rat adenosine A1 receptor initiated by stimulation with the full agonist 2-chloro-N6-cyclopentyladenosine (CCPA) and by six structurally distinct partial agonists of this receptor was investigated. Endogenous G protein alpha subunits in rat cortical membranes were inactivated by N-ethylmaleimide (NEM). Activation of rat recombinant myristoylated alpha(o), alpha(i1), alpha(i2) and alpha(i3) by partial agonists in comparison to the full agonist was assessed by guanosine-5'-(gamma-[35S]thio)triphosphate ([35S]GTPgammaS) binding after reconstitution of G protein alpha subunits with the adenosine A1 receptor in N-ethylmaleimide-treated membranes. 2-Chloro-N6-cyclopentyladenosine and 3' -deoxy-N6-cyclopentyladenosine (3'-d-CPA), the partial agonist with the highest intrinsic activity, were significantly more potent in activation of alpha(i) subtypes than alpha(o). In contrast, 5'-methylthioadenosine (MeSA), 2'-deoxy-2-chloroadenosine (cladribine), 2'-deoxy-N6-cyclopentyladenosine (2'-d-CPA), 2-phenylaminoadenosine (CV 1808) and C8-aminopropyl-N6-cyclopentyladenosine (C8-aminopropyl-CPA) did not exhibit higher potency for Go or any Gi subtype. All partial agonists, although carrying structurally different modifications, showed higher relative intrinsic activities in activation of Gi than of Go, indicating that Gi-coupled pathways may be activated selectively via the A1 receptor by partial agonists, but not Go-mediated responses.     

G proteins of the Gq family couple the H2 histamine receptor to phospholipase C

In several cell systems histamine has been shown to stimulate both adenylyl cyclase and phospholipase C through activation of a G protein-coupled H2 receptor. To analyze the bifurcating signal emanating from the activated H2 receptor and to identify the G proteins involved, H1 and H2 histamine receptors were functionally expressed in baculovirus-infected insect cells. Histamine challenge lead to concentration-dependent cAMP formation and Ca2+ mobilization in Sf9 cells infected with a virus encoding the H2 receptor, whereas H1 receptor stimulation only resulted in pronounced phospholipase C activation. To analyze the G protein coupling pattern of histamine receptors, activated G proteins were labeled with [alpha-32P]GTP azidoanilide and identified by selective immunoprecipitation. In insect cell membranes expressing H1 histamine receptors, histamine led to incorporation of the label into alpha q-like proteins, whereas activation of the H2 receptor resulted in labeling of alpha q- and alpha s-like G protein alpha-subunits. In COS cells transfected with H2 receptor complementary DNA, histamine caused concentration-dependent accumulation of cAMP and inositol phosphates; the latter effect was insensitive to pertussis toxin treatment. Histamine stimulation led to a pronounced increase in inositol phosphate production when complementary DNAs coding for alpha q, alpha 11, alpha 14, or alpha 15 G protein alpha-subunits were cotransfected. This increase was specific for Gq family members, as overexpression of alpha 12 or alpha s did not enhance histamine-stimulated phospholipase C activation. In membranes of guinea pig heart, addition of [alpha-32P]GTP azidoanilide resulted in labeling of alpha q and alpha 11 via the activated H1 and also via H2 receptors. These data demonstrate that dual signaling of the activated H2 histamine receptor is mediated by coupling of the receptor to Gs and Gq family members.     

In vivo coupling of insulin-like growth factor II/mannose 6-phosphate receptor to heteromeric G proteins. Distinct roles of cytoplasmic domains and signal sequestration by the receptor

We examined the signaling function of the IGF-II/mannose 6-phosphate receptor (IGF-IIR) by transfecting IGF-IIR cDNAs into COS cells, where adenylyl cyclase (AC) was inhibited by transfection of constitutively activated G alpha i cDNA (G alpha i2Q205L). In cells transfected with IGF-IIR cDNA, IGF-II decreased cAMP accumulation promoted by cholera toxin or forskolin. This effect of IGF-II was not observed in untransfected cells or in cells transfected with IGF-IIRs lacking Arg2410-Lys2423. Thus, IGF-IIR, through its cytoplasmic domain, mediates the Gi-linked action of IGF-II in living cells. We also found that IGF-IIR truncated with C-terminal 28 residues after Ser2424 caused G beta gamma-dominant response of AC in response to IGF-II by activating Gi. Comparison with the G alpha i-dominant response of AC by intact IGF-IIR suggests that the C-terminal 28-residue region inactivates G beta gamma. This study not only provides further evidence that IGF-IIR has IGF-II-dependent signaling function to interact with heteromeric G proteins with distinct roles by different cytoplasmic domains, it also suggests that IGF-IIR can separate and sequestrate the G alpha and G beta gamma signals following Gi activation.     

Differential coupling of alpha1-, alpha2-, and beta-adrenergic receptors to mitogen-activated protein kinase pathways and differentiation in transfected PC12 cells

Three adrenergic receptor families that selectively activate three different G proteins (alpha1/Gq/11, alpha2/Gi, and beta/Gs) were used to study mitogen-activated protein kinase (MAPK) activation and differentiation in PC12 cells. PC12 cells were stably transfected with alpha1A-, alpha2A-, or beta1-adrenergic receptors (ARs) in an inducible expression vector, and subclones were characterized. Norepinephrine stimulated inositol phosphate formation in alpha1A-transfected cells, inhibited cyclic adenosine 3'5'-monophosphate (cAMP) formation in alpha2A-transfected cells, and stimulated cAMP formation in beta1-transfected cells. Nerve growth factor activated extracellular signal-regulated kinases (ERKs) in all cell lines; however, norepinephrine activated ERKs only in alpha1A- and beta1-transfected cells but not in alpha2A-transfected cells. Norepinephrine also activated c-Jun NH2-terminal kinase and p38 MAPK in alpha1A-transfected cells but not in beta1- or alpha2A-transfected cells. Norepinephrine caused differentiation of PC12 cells expressing alpha1A-ARs but not those expressing beta1- or alpha2A-ARs. However, norepinephrine acted synergistically with nerve growth factor in promoting differentiation of cells expressing beta1-ARs. Whereas ERKs are activated by Gi- but not Gs-linked receptors in many fibroblastic cell lines, we observed the opposite in PC12 cells. The results show that activation of the different G protein signaling pathways has different effects on MAPKs and differentiation in PC12 cells, with Gq signaling pathways activating all three major MAPK pathways.     

Differential intracellular signaling of the GalR1 and GalR2 galanin receptor subtypes

The diverse physiological functions exerted by the neuropeptide galanin may be regulated by multiple G protein-coupled receptor subtypes and intracellular signaling pathways. Three galanin receptor subtypes (GalRs) have been recently cloned, but the G protein coupling profiles of these receptors are not completely understood. We have generated GalR1- and GalR2-expressing Chinese hamster ovary (CHO) cell lines and systematically examined the potential for these two receptors to couple to the Gs, Gi, Go, and Gq proteins. Galanin did not stimulate an increase in cAMP levels in GalR1/CHO or GalR2/CHO cells, suggesting an inability of either receptor to couple to Gs. Galanin inhibited forskolin-stimulated cAMP production in GalR1/CHO cells by 70% and in GalR2/CHO cells by 30%, suggesting a strong coupling of GalR1 to Gi and a more modest coupling between GalR2 and Gi. GalR1 and GalR2 both mediated pertussis toxin-sensitive MAPK activity (2-3-fold). The stimulation mediated by GalR1 was inhibited by expression of the C-terminus of beta-adrenergic receptor kinase (beta ARKct), which specifically inhibits G beta gamma signaling, but was not affected by the protein kinase C (PKC) inhibitor, bis[indolylmaleimide], or cellular depletion of PKC. In contrast, GalR2-mediated MAPK activation was not affected by beta ARKct expression but was abolished by inhibition of PKC activity. The data demonstrate that GalR1 is coupled to a Gibetagamma signaling pathway to mediate MAPK activation. In contrast, GalR2 utilizes a distinct signaling pathway to mediate MAPK activation, which is consistent with Go-mediated MAPK activation in CHO cells. Galanin was unable to stimulate inositol phosphate (IP) accumulation in CHO or COS-7 cells expressing GalR1. In contrast, galanin stimulated a 7-fold increase in IP production in CHO or COS-7 cells expressing GalR2. The GalR2-mediated IP production was not affected by pertussis toxin, suggesting a linkage of GalR2 with Gq/G11. Thus, the GalR1 receptor appears to activate only the Gi pathway. By contrast, GalR2 is capable of stimulating signaling which is consistent with activation of Go, Gq/G11, and Gi. The differential signaling profiles and the tissue distribution patterns of GalR1 and GalR2 may underlie the functional spectra of galanin action mediated by these galanin receptors and regulate the diverse physiological functions of galanin.     

Glucocorticoid regulation of G-protein subunits in neonatal liver

The effect of dexamethasone administration in vivo on the steady-state levels of G-protein subunits in liver of neonatal rabbits was investigated using specific antibodies to each subunit as well as bacterial toxin-mediated ADP-ribosylation assays. Parallel measurements were also made of the activity of adenylyl cyclase, as influenced by a variety of activators. Dexamethasone administration modulated the levels of G-protein subunits in liver in an age-dependent and subunit-specific manner but not in 24-h-old newborns. The inductive effect of dexamethasone was observed in animals older than 24 h, the greatest effect being on 2- to 3-day-old neonates. In 48-h-old animals the alpha-subunits Gs alpha-1, Gs alpha-2, Gi alpha and the beta-subunit G beta increased 2.0-, 2.1-, 4.3- and 2.8-fold, respectively, compared to the control. The increases were much less for older animals. Dexamethasone treatment also modulated effector-mediated stimulation of adenylyl cyclase activity in vitro and mimicked its effects on G-protein levels; the greatest increase (approximately 2-fold) in the activation of adenylyl cyclase occurred in membranes isolated from 2- to 3-day-old animals. In older animals there was either no effect of dexamethasone or a decrease in activity. The degree of change in enzyme activity paralleled the change in the amount of Gs alpha rather than of Gi alpha or G beta. These results suggest development-dependent regulation of hepatic G-proteins by glucocorticoids.     

Guanine nucleotide-binding inhibitory protein-mediated inhibition of adenylyl cyclase is enhanced in spontaneously hypertensive rat preglomerular arteriolar smooth muscle cells

The purpose of our study was to determine whether Gi-mediated control over adenylyl cyclase in preglomerular arteriolar smooth muscle cells (PGASMC) is enhanced in the spontaneously hypertensive rat (SHR). PGASMC were cultured from preglomerular microvessels isolated from adult SHR (14-15 wk of age) and age-matched WKY rats. Confluent monolayers of cells in third passage were used for the experiments. cAMP released into the media (30 min) as well as cellular levels of cAMP were measured in the presence of a phosphodiesterase inhibitor, 1-isobutyl-3-methyl-xanthine (IBMX; 100 microM) and expressed as pmol/mg protein. Total (released + cellular) cAMP was significantly lower in SHR (14.19 +/- 2.30 pmol/mg protein) as compared with WKY (28.3 +/- 3.04 pmol/mg protein). Correspondingly, the released (4.6 +/- 0.4 pmol/mg protein) as well as cellular (9.78 +/- 2.18 pmol/mg protein) cAMP levels were also significantly lower in SHR when compared with WKY (8.85 +/- 1.26 and 18.86 +/- 2.0 pmol/mg protein, respectively). The steady-state levels of none of the Gi alpha subunits, namely Gi alpha 1, Gi alpha 2 and Gi alpha 3, were higher in the SHR PGASMC. Pertussis toxin treatment (PTX; 100 ng/ml; 24 hr) caused complete ADP-ribosylation of Gi alpha subunits in both WKY and SHR PGASMC. The same treatment of PTX also produced a significant increase in total cAMP in SHR, but not in WKY, such that the total cAMP levels after PTX treatment were not significantly different between the two strains. Interestingly, PTX significantly increased the released (20.26 +/- 0.90 pmol/mg protein) but not the cellular (13.63 +/- 1.63 pmol/mg protein) cAMP in SHR. Forskolin (1 microM) induced similar increases in total cAMP and isoproterenol (1 microM) caused greater increases in total cAMP in SHR cells compared with WKY cells. These data strongly suggest that in SHR PGASMC total adenylyl cyclase activity is not altered. Furthermore, steady-state expressions of Gi alpha-1, Gi alpha-2 and Gi alpha-3 are not increased whereas Gi-mediated inhibition of adenylyl cyclase is augmented in SHR PGASMC.     

Gi-mediated activation of the p21ras-mitogen-activated protein kinase pathway by alpha 2-adrenergic receptors expressed in fibroblasts

The alpha 2-adrenergic receptors are linked to inhibition of adenylylcyclase and, under certain circumstances, to stimulation of phospholipid hydrolysis via pertussis toxin-sensitive G proteins. Here we show that alpha 2-adrenergic receptors can couple to an alternative signaling pathway. When expressed in Rat-1 cells, stimulation of the alpha 2A receptor, which couples to Gi2 and Gi3, causes rapid, transient activation of the protooncogene product p21ras as measured by an increase in the amount of bound GTP. Furthermore, alpha 2A receptor stimulation causes rapid phosphorylation of the p42 mitogen-activated protein (MAP) kinase. Pertussis toxin completely inhibits both p21ras activation and MAP kinase phosphorylation, but both responses appear to be independent of adenylylcyclase inhibition or phospholipase stimulation. Thus, alpha 2-adrenergic receptors can couple to the p21ras-MAP kinase pathway via Gi, which may explain the mitogenic potential of alpha 2 agonists in certain cell types; together with previous results, these findings further suggest that activation of this pivotal signaling pathway may be a common event in the action of Gi-coupled receptors.     

G protein Gi2 alpha in the cochlea: cloning and selective occurrence in receptor cells

A cDNA library was made from the mouse cochlea and screened with a G protein-cDNA like molecule obtained from cochlear tissue by polymerase chain reaction. The nucleotide sequence of a clone, named cochlear Gi2 alpha, had 99.2% identity to mouse macrophage Gi2 alpha. Using an antibody which is selective for Gi2 alpha, expression of the cochlear Gi2 alpha was localized in outer and inner hair cells of the organ of Corti. Possible functional roles of this G protein in hair cells are discussed.     

Beta-adrenergic signal transduction in the failing and hypertrophied myocardium

A strong sympathetic activation has been observed in heart failure and is the cause of beta-adrenergic desensitization in this condition. On the receptor level there is downregulation of beta1-adrenergic receptors and uncoupling of beta2-adrenoceptors. The latter mechanism has been related to an increased activity and gene expression of beta-adrenoceptor kinase in failing myocardium, leading to phosphorylation and uncoupling of receptors. beta3-Adrenoceptors mediate negative inotropic effects, but alterations in these receptors are not known. In addition, an increase in inhibitory G protein alpha subunits (Gi alpha) has been suggested to be causally linked to adenylyl cyclase desensitization in heart failure. In contrast, the catalytic subunit of adenylyl cyclase, stimulatory G protein alpha and betagamma subunits, have been observed to be unchanged. Recent evidence shows that increases in Gi alpha also depress adenylyl cyclase in compensated cardiac hypertrophy both in monogenic and polygenic and in secondary hypertension. These increases of Gi alpha can suppress adenylyl cyclase in the absence of beta-adrenergic receptor downregulation. Since cardiac hypertrophy in pressure overload is a strong predictor of cardiac failure, these observations indicate that adenylyl cyclase desensitization by Gi alpha may be a pathophysiologically relevant mechanism contributing to the progression from compensated cardiac hypertrophy to heart failure.