GTP-binding membrane proteins in activated and differentiating T cells

We have earlier reported changes in the GTP binding of several membrane proteins including Gs alpha and Gi alpha during thymic differentiation of T cells. Using an [alpha-32P]GTP-photoaffinity labeling technique we have studied the pattern of GTP binding proteins in activated and resting T lymphocytes and in T cells induced to differentiate by TPA. The GTP binding proteins in mitogen-activated T cells resembled those seen in leukemia T cell lines. Treatment of Jurkat, but not of CCRF-CEM, T cells with TPA caused increased GTP-labeling of a 34 kDa protein and Gi alpha. The GTP labeling pattern in TPA-treated Jurkat cells resembled that in resting T lymphocytes. TPA induced de novo expression of functional TCR/CD3 on CCRF-CEM and downregulation of TCR/CD3 on Jurkat cells but these changes did not correlate with the altered GTP-labeling patterns.     

Factors determining the specificity of signal transduction by guanine nucleotide-binding protein-coupled receptors. I. Coupling of alpha 2-adrenergic receptor subtypes to distinct G-proteins.

alpha 2-Adrenergic receptor (alpha 2-AR) subtypes couple to pertussis toxin (PT)-sensitive G-proteins to elicit both stimulatory and inhibitory cell responses. Signal specificity may be generated by the ability of the receptor subtypes to "recognize" distinct G-proteins with different affinity. To address this issue we stably expressed three alpha 2-AR subtypes, RNG alpha 2 (alpha 2B-AR), RG10 (alpha 2C-AR), and RG20 (alpha 2D-AR), in NIH-3T3 fibroblasts, which express two PT-sensitive G-proteins (Gi alpha 2, Gi alpha 3), and analyzed receptor/G-protein interactions by determining: 1) functional coupling to adenylylcyclase and 2) the ability of the receptors to exist in a high affinity state for agonist. In alpha 2D-AR transfectants expressing 200 or 2,200 fmol of receptor/mg of protein, epinephrine (10 microM) inhibited forskolin-induced elevation of cellular cAMP by 26 +/- 4.8% and 72 +/- 6.2%, respectively. Similar results were obtained in alpha 2B-AR transfectants. However, in alpha 2C-AR transfectants (200 fmol/mg) the forskolin-induced elevation of cellular cAMP was not altered by agonist treatment. In alpha 2C-AR transfectants expressing higher receptor densities (650-1,200 fmol/mg), epinephrine inhibited the effect of forskolin by 30 +/- 3.2%. This difference in functional coupling among the alpha 2-AR subtypes is reflected at the receptor/G-protein interface. In membrane preparations of alpha 2B and alpha 2D-AR but not alpha 2C-AR transfectants, agonist competition curves were biphasic, indicating high and low affinity states of the receptor for agonist. The high affinity state was guanyl-5'-yl imidodiphosphate- and PT-sensitive, indicative of receptor/G-protein coupling. These data suggest that the alpha 2C-AR differs from the alpha 2B and alpha 2D-AR subtypes in its ability to recognize PT-sensitive G-proteins expressed in NIH-3T3 fibroblasts. The alpha 2C-AR may couple preferentially to PT-sensitive G-proteins (Gi1, Go1,2) not expressed in NIH-3T3 fibroblasts and thereby elicit different cellular responses.     

Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

Prostaglandin E2 (PGE2) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet (Negishi, M., Ito, S., Tanaka, T., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1987) J. Biol. Chem. 262, 12077-12084). In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, we purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its alpha subunit from two known pertussis toxin substrate G-proteins (Gi and Go) purified from bovine brain. The molecular weight of the alpha subunit was 40,000, which is between those of Gi and Go. The purified protein was also distinguished immunologically from Gi and Go and was referred to as Gam. PGE receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and freed from G-proteins by wheat germ agglutinin column chromatography. Reconstitution of the PGE receptor with pure Gam, Gi, or Go in phospholipid vesicles resulted in a remarkable restoration of [3H]PGE2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. The displacement of [3H]PGE2 binding was specific for PGE1 and PGE2. Furthermore, addition of PGE2 stimulated the GTPase activity of the G-proteins in reconstituted vesicles. These results indicate that the PGE receptor can couple functionally with Gam, Gi, or Go in phospholipid vesicles and suggest that Gam may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.     

Effects of modulators of adenylyl cyclase on interleukin-2 production, cytosolic Ca2+ elevation, and K+ channel activity in Jurkat T cells

We have studied the effects of prostaglandin E2 (PGE2) and cholera toxin, two modulators of adenylyl cyclase, and 8-bromo cAMP (8-BrcAMP) on various parameters of lymphocyte activation using the human T cell line Jurkat. Our results show that PGE2 and cholera toxin inhibit, in a dose-related manner, the phytohemagglutinin (PHA)-dependent production of interleukin 2 by these cells. The data are consistent with the interpretation that the inhibition is due to an intracellular increase in cAMP, since the metabolically stable 8-BrcAMP analog produced the same inhibitory effect. However, PGE2 or 8-BrcAMP did not interfere with the PHA-induced elevation in the cytosolic concentration of Ca2+, suggesting that changes in the intracellular concentration of cAMP does not affect the internal release or the influx of Ca2+. In contrast, cholera toxin prevented the Ca2+ response of Jurkat cells to PHA. We studied the effects of PGE2, cholera toxin, and 8-BrcAMP on the amplitude of the K+ outward current using the patch clamp technique in the whole cell configuration. Results showed that PGE2, 8-BrcAMP, and cholera toxin inhibited K+ channel activity. For instance, the amplitude of the outward K+ current was reduced to 43 +/- 19%, 50 +/- 26%, and 46 +/- 16% of control values in the case of cells perfused in the presence of PGE2, 8-BrcAMP, and cholera toxin, respectively. Blocking K+ channels with tetraethylammonium ions did not prevent the characteristic Jurkat Ca2+ response to PHA. Our observations that cAMP inhibits K+ channel activity in a T cell line provide an additional explanation for its reported inhibition of lymphocyte activation. Increasing the intracellular concentration of cAMP may result in reduction of K+ movements and in negative modulation of signal transduction via G-proteins as previously suggested. These two effects could act in synergy to impair signal transduction.     

Delta-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2.

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.     

Ethanol differentially regulates G proteins in neural cells

Long-term incubation of clonal neural cell lines with ethanol differentially reduces the stimulation of cAMP accumulation by hormones and cholera toxin. In the NG108-15 neuroblastoma chi glioma hybrid cell line, this heterologous desensitization was associated with a 42% reduction in the expression of Gs alpha and no significant change in Gi alpha. By contrast, ethanol treatment of the parental neuroblastoma cell line N18TG2 caused little loss of response to hormones or cholera toxin and no significant change in Gs alpha or Gi alpha. Ethanol induced heterologous desensitization in N1E-115 neuroblastoma cells; however, this cell line showed a dose-dependent increase in Gi alpha and a later decrease in Gs alpha. Thus, ethanol causes heterologous desensitization of hormone-stimulated cAMP accumulation by different mechanisms in related neural cell lines.     

Purification of the major GTP-binding proteins from human placental membranes

Using minor modifications of procedures developed to purify GTP-binding proteins (G-proteins) from rabbit liver, we have purified the major G-proteins present in human placental membranes. One, referred to as Gi, is the major substrate for pertussis toxin-catalyzed ADP-ribosylation and has an alpha-subunit of 41,000 daltons, and beta-subunit of 36,000 and 35,000 daltons, and a gamma-subunit of 10,000 daltons. The other protein, referred to as Gp, was identified by its ability to bind guanine nucleotides specifically with high affinity. This activity was resolved from Gs and Gi by the second step of purification (AcA-34 chromatography) and was further purified through heptylamine-Sepharose and hydroxylapatite. The guanine nucleotide-binding site, which can be resolved by high performance liquid chromatography procedures and identified by a photolyzable GTP analogue, is associated with a 21,000-dalton protein (Gp alpha) that copurifies with beta gamma-subunits indistinguishable from the beta gamma-subunits associated with Gs and Gi. This protein represents a potentially novel member of the structurally and functionally homologous family of G-proteins that are transducing elements in the receptor-mediated regulation of a variety of cellular processes.     

Simultaneous coupling of alpha 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of alpha 2C10, alpha 2C4, and alpha 2C2 adrenergic receptors to Gi and Gs.

Coupling of the three alpha 2-adrenergic receptor (alpha 2AR) subtypes to Gi and Gs was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the alpha 2AR agonist UK-14304, alpha 2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, alpha 2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of alpha 2C10 to both Gi and Gs. To isolate functional Gs and Gi coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only alpha 2C10-mediated inhibition (approximately 50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only alpha 2C10-mediated stimulation (approximately 60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl-terminal portion of Gs alpha blocked this functional alpha 2AR.Gs coupling to the same extent as that found for beta 2AR.Gs coupling. In addition to functional Gs coupling, we also verified direct, agonist-dependent, physical coupling of alpha 2AR to Gs alpha. In agonist-treated membranes, an agonist-receptor-Gs alpha complex was immunoprecipitated with a specific alpha 2C10 antibody, and the Gs component identified by both western blots using Gs alpha antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the alpha 2AR subtypes, we investigated whether G-protein coupling was subtype-selective, using UK-14304 and cells with the same alpha 2AR expression levels (approximately 5 pmol/mg). Coupling to Gi was equivalent for alpha 2C10, alpha 2C4, and alpha 2C2: 53.4 +/- 8.8% versus 54.9 +/- 1.0% versus 47.6 +/- 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to Gs were found between the three alpha 2AR subtypes: stimulation of adenylyl cyclase was 57.9 +/- 6.3% versus 30.7 +/- 1.1% versus 21.8 +/- 1.7% for alpha 2C10, alpha 2C4, and alpha 2C2, respectively. Thus, alpha 2AR have the potential to couple physically and functionally to both Gi and Gs; for Gi coupling we found a rank order of alpha 2C10 = alpha 2C4 = alpha 2C2, while for Gs coupling, alpha 2C10 greater than alpha 2C4 greater than alpha 2C2.     

Localization of mRNAs encoding the alpha-subunits of signal-transducing G-proteins within rat brain and among peripheral tissues

The sequence of the mRNAs which encode the alpha-subunits of the signal-transducing G-proteins Gs, Go and two forms of Gi (termed Gi1 and Gi2) have recently been reported. Based on rat sequences we prepared oligodeoxynucleotide probes for measurement of these mRNAs in rat brain and peripheral tissues. The relative abundance of these mRNA species in brain was Gs greater than Go approximately Gi2 greater than Gi1. The Gs and Gi2 mRNAs had somewhat lower levels in heart, kidney and liver than in brain, and Go and Gi1 mRNAs were not detected in the peripheral tissues. Using in situ hybridization we localized each of these mRNAs within slices of the rat brain. The patterns of distribution of Gs and Gi2 mRNA were very similar, but very different from that of Go and Gi1 mRNA. These data illustrate that receptor-effector coupling G-proteins are regionally specialized in their expression. This regional specialization may reflect a selective coupling of individual G-proteins with the various neurotransmitter receptors and effector pathways.     

Alterations in mRNA levels, expression, and function of GTP-binding regulatory proteins in adipocytes from obese mice (C57BL/6J-ob/ob)

Messenger RNA levels for the alpha subunit of G-proteins expressed in adipocytes of lean and obese (ob/ob) mice were compared with relative levels of the encoded proteins. Using both toxin labeling and Western blots, expression of Gs alpha, Gi alpha-1, and Gi alpha-3 was decreased by approximately 2-fold in adipocytes of obese mice, while levels of Gi alpha-2 did not differ between the phenotypes. The decreases in Gi alpha-1 and Gs alpha in the obese mouse were attributed to decreased mRNA levels for these proteins. Similar mRNA levels for Gi alpha-3 were noted in both phenotypes, but Gi alpha-2 message was increased 2-fold in the obese mouse. Inhibitory regulation of adipocyte adenylylcyclase through G-proteins was evaluated by comparing the ability of R-PIA to inhibit isoproterenol-stimulated responses between the phenotypes. In spite of the decrease in Gi alpha-1 and Gi alpha-3 in adipocytes from obese mice, R-PIA inhibited adenylylcyclase, cAMP-dependent protein kinase, and lipolysis in similar fashion in both phenotypes. The GTP analog, Gpp(NH)p also inhibited forskolin-stimulated adenylylcyclase in a comparable manner, but the magnitude of the inhibition was slightly less in adipocyte membranes from obese mice. In contrast, the decrease in expression of Gs alpha was translated into substantially poorer activation of isoproterenol-stimulated responses in the obese mouse. The concentration of isoproterenol producing half-maximal activation of adenylylcyclase, protein kinase, and lipolysis did not differ between the phenotypes, but the maximal responses were much lower in cells from obese mice. Similar lipolytic potential in isolated adipocytes from each phenotype and similar total forskolin-stimulated cyclase activity in adipocyte membranes from each phenotype suggest that decreased expression of Gs alpha may contribute to the characteristic alteration in mobilization of triglycerides noted in adipocytes from obese mice.     

G-protein mRNA levels during adipocyte differentiation

G-protein-mediated transmembrane signaling in 3T3-L1 cells is modulated by differentiation. The regulation of G-protein expression in differentiating 3T3-L1 cells was probed at the level of mRNA by DNA-excess solution hybridization. Pertussis toxin-catalyzed ADP-ribosylation of G-protein alpha-subunits increased as fibroblasts differentiate to adipocytes. Steady-state levels of mRNA for Gi alpha 2 and Go alpha, in contrast, declined sharply. Immunoblotting with antipeptide antibodies specific for Gi alpha 2, too, revealed a decline in the steady-state expression of this pertussis toxin substrate. ADP-ribosylation of Gs alpha by cholera toxin was less in the adipocyte than fibroblast. Analysis by immunoblotting revealed only a modest decline in Gs alpha. Analysis of mRNA levels also demonstrated a decline for Gs alpha. mRNA levels for the G beta-subunits rose initially (25%) on day 1, declined from day 1 to day 3, and remained 25% lower in adipocytes than in fibroblasts. In 3T3-L1 adipocytes the molar amounts of subunit mRNAs were: 60.6 (Gs alpha); 2.1 (Gi alpha 2); and 1.5 (Go alpha) amol/microgram total cellular RNA. In rat fat cells these mRNA levels were 19.4 (Gs alpha); 7.0 (Gi alpha 2); and 2.3 (Go alpha). These data demonstrate that for Gi alpha 2 and Go alpha alike mRNA and protein expression decrease, not increase, in differentiation. A substrate for pertussis toxin other than Gi alpha 2 and Go alpha appears to be responsible for the increase in toxin-catalyzed labeling that accompanies differentiation of 3T3-L1 cells.     

Prostaglandin E2 inhibits the activation of cloned T cell hybridomas

To minimize complicating interactions inherent in heterogeneous cell populations, we used a panel of cloned murine autoreactive (E8.A1) and antigen-specific (HEL.C10, HEL.B14) T cell hybridomas to examine the effect of prostaglandin E2 (PGE2) on T cell activation. These T cells secrete interleukin 2 (IL 2) when co-cultured with a cloned population of I region-matched stimulator cells (TA3), or with mitogenic signals in the absence of TA3 stimulator cells. Physiologic concentrations of PGE2 inhibited the induction of IL 2 secretion by the T cell hybridomas tested, when they were activated either by TA3 cells or by mitogenic signals. IL 2 production was inhibited in a dose-dependent manner by concentrations of PGE2 between 10(-7) and 10(-11) M, with 50% inhibition occurring at 10(-10) M. Pretreatment of the T hybridoma cells with 10(-7) M PGE2 for 1 hr before culture also resulted in marked inhibition of IL 2 secretion. Similar pretreatment of the TA3 cells did not affect their ability to activate the T cell hybridomas. PGE2 at 10(-8) M induced a 30-fold increase in cAMP levels within 25 min of addition to culture of the E8.A1 T cell hybridoma, but caused no significant elevation of cAMP levels in TA3 cells. The direct addition of dibutyryl cAMP (dcAMP) to cultures of E8.A1 cells resulted in marked inhibition of IL 2 secretion when stimulated by TA3 or by mitogenic signals, with an average of 80% inhibition occurring at 10(-4) M dcAMP. PGE2 and dcAMP also inhibited the growth of E8.A1 cells. Initially, cell growth was virtually halted, but began to recover between 24 and 48 hr after the addition of either PGE2 or dcAMP. Neither PGE2 nor dcAMP inhibited the division of TA3 cells. High affinity binding sites for PGE2 were detected in the E8.A1 T cell hybridomas with an apparent Kd of 7.6 X 10(-10) M, which is consistent with the functional data. No specific binding was detected in the TA3 stimulator cells. These findings suggest that the immunosuppressive effects of PGE2 are localized to the T cell, are receptor regulated, and may be mediated by the associated increase of cAMP levels in the T cell hybridomas.     

Regulation of prostaglandin E2 binding to a murine macrophage cell line, P388D1, by insulin.

Preincubation of murine macrophage-like P388D1 cells with physiological amounts of insulin resulted in an increase in prostaglandin E2 binding to these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0.30 +/- 0.02 to 0.63 +/- 0.03 fmol/10(6) cells for the high affinity receptor binding sites, and from 2.4 +/- 0.31 to 5.0 +/- 0.41 fmol/10(6) cells for the low affinity receptor binding sites) rather than to an increase in the affinity of the binding sites. The insulin-stimulation of PGE2 binding appeared to be associated with a lowering of the cAMP level in these cells; treatment of cells with insulin lowered the cAMP level by increasing the cAMP phosphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressive action of this prostanoid, as PGE2 is known to suppress many steps in the immune response, including interleukin-1 expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiological concentrations may enhance the immunosuppressive action of PGE2.     

Prostaglandin E2 can bimodally inhibit and stimulate the epididymal adipocyte adenylyl cyclase activity.

Measurements of prostaglandin E2 (PGE2)-induced adenylyl cyclase activity in membranes isolated from epididymal rat adipocytes revealed inhibition of cAMP production at low concentrations of PGE2 (less than 10 mM) and stimulation at higher concentrations. This biphasic effect of PGE2 was obtained when adenylyl cyclase was stimulated with GTP or NaF. In the presence of forskolin only the inhibitory phase by PGE2 was observed. Sulprostone, a PGE2 analogue, did not affect cAMP synthesis in the presence of either GTP or NaF; however, in the presence of forskolin, it inhibited cAMP production similarly to PGE2. Treatment of the membranes with cholera or pertussis toxin did not alter the biphasic effect of PGE2 on cAMP production. These findings raise the possibility that PGE2 acts through several receptor subtypes which are coupled to GTP binding proteins different from the classical Gi or Gs proteins.     

Cross-regulation between G-protein-mediated pathways. Stimulation of adenylyl cyclase increases expression of the inhibitory G-protein, Gi alpha 2

The hormone-sensitive adenylyl cyclase system is under dual control, receiving both stimulatory and inhibitory inputs. Guanine nucleotide-binding regulatory proteins (G-proteins) transduce signals from cell surface receptors to effectors such as adenylyl cyclase. Hormonal stimulation is propagated via Gs, inhibition by Gi. Persistent (24-h) activation of the stimulatory pathway of adenylyl cyclase by the diterpene forskolin or the beta-adrenergic agonist isoproterenol in S49 mouse lymphoma cells enhanced the effects of somatostatin mediated via the inhibitory pathway of adenylyl cyclase. Stimulating cells with forskolin or isoproterenol for 24 h resulted in a 3-fold increase in the steady-state levels of Gi alpha 2 and a 25% decline in Gs alpha, as quantified by immunoblotting. Within 12 h of stimulation of adenylyl cyclase, Gi alpha 2 mRNA levels increased 4-fold, measured by DNA-excess solution hybridization. Gs alpha mRNA levels, in contrast, increased initially (25%), but then declined to 75% of control. In S49 variants that lack functional protein kinase A (kin-), stimulation by isoproterenol failed to alter Gi alpha 2 expression at either the protein or the mRNA levels. A 3-fold increase in relative synthesis rate and no change in the half-life (approximately 80 h) of Gi alpha 2 was observed in response to forskolin stimulation. Although Gs alpha synthesis increased (70%) modestly in response to forskolin stimulation, the half-life of Gs alpha actually decreased from 55 h in naive cells to 34 h in treated cells. Thus, the two G-protein-mediated pathways controlling adenylyl cyclase display "cross-regulation." Persistent activation of the stimulatory pathway increases Gi alpha 2 mRNA and expression. Transiently elevated Gs alpha mRNA levels are counterbalanced by a reduction in the half-life of the protein.     

Principles and Determinants of G-Protein Coupling by the Rhodopsin-Like Thyrotropin Receptor

In this study we wanted to gain insights into selectivity mechanisms between G-protein-coupled receptors (GPCR) and different subtypes of G-proteins. The thyrotropin receptor (TSHR) binds G-proteins promiscuously and activates both Gs (cAMP) and Gq (IP). Our goal was to dissect selectivity patterns for both pathways in the intracellular region of this receptor. We were particularly interested in the participation of poorly investigated receptor parts.We systematically investigated the amino acids of intracellular loop (ICL) 1 and helix 8 using site-directed mutagenesis alongside characterization of cAMP and IP accumulation. This approach was guided by a homology model of activated TSHR in complex with heterotrimeric Gq, using the X-ray structure of opsin with a bound G-protein peptide as a structural template.We provide evidence that ICL1 is significantly involved in G-protein activation and our model suggests potential interactions with subunits Gα as well as Gβγ. Several amino acid substitutions impaired both IP and cAMP accumulation. Moreover, we found a few residues in ICL1 (L440, T441, H443) and helix 8 (R687) that are sensitive for Gq but not for Gs activation. Conversely, not even one residue was found that selectively affects cAMP accumulation only.Together with our previous mutagenesis data on ICL2 and ICL3 we provide here the first systematically completed map of potential interfaces between TSHR and heterotrimeric G-protein. The TSHR/Gq-heterotrimer complex is characterized by more selective interactions than the TSHR/Gs complex. In fact the receptor interface for binding Gs is a subset of that for Gq and we postulate that this may be true for other GPCRs coupling these G-proteins. Our findings support that G-protein coupling and preference is dominated by specific structural features at the intracellular region of the activated GPCR but is completed by additional complementary recognition patterns between receptor and G-protein subtypes.     

G-protein subunit mRNA levels in rat heart, liver, and adipose tissues: analysis by DNA-excess solution hybridization

The steady-state levels of mRNAs for the G-proteins Gi alpha 2, Go alpha, and the G beta-subunits common to each were established in rat adipose, heart and liver. Uniformly-radiolabeled, single-stranded antisense probes were constructed from cDNAs or assembled from oligonucleotides. Direct comparison of the steady-state levels of the G-protein mRNAs was performed under identical assay conditions, and on a molar basis. In adipose, liver and heart, Gs alpha mRNA was more abundant than mRNA for Go alpha, Gi alpha, and G beta. In adipose tissue, mRNA levels were as follows: 19.4, 7.6, 7.0, and 2.3 amol mRNA per micrograms total cellular RNA for Gs alpha, G beta, Gi alpha 2, and Go alpha, respectively. In heart Gs alpha mRNA was less abundant than in adipose, but the relative trend among the G-protein subunits was the same. In liver, G beta mRNA was more abundant than either Go alpha or Gi alpha 2. Go alpha mRNA levels ranged from 1.2 to 2.3 amol/micrograms total RNA in liver and adipose, respectively. The present work demonstrates the many advantages of this strategy when applied to the study of a family of homologous, low-abundance proteins and establishes for the first time the molar levels of Gi alpha 2, Gs alpha, Go alpha, and G beta-subunit mRNAs in several mammalian tissues.     

The beta3-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a Gi-dependent mechanism

Promiscuous coupling between G protein-coupled receptors and multiple species of heterotrimeric G proteins provides a potential mechanism for expanding the diversity of G protein-coupled receptor signaling. We have examined the mechanism and functional consequences of dual Gs/Gi protein coupling of the beta3-adrenergic receptor (beta3AR) in 3T3-F442A adipocytes. The beta3AR selective agonist disodium (R, R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1, 3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in cAMP production in adipocyte plasma membrane preparations, and pretreatment of cells with pertussis toxin resulted in a further 2-fold increase in cAMP production by CL316,243. CL316,243 (5 microM) stimulated the incorporation of 8-azido-[32P]GTP into Galphas (1.57 +/- 0.12; n = 3) and Galphai (1. 68 +/- 0.13; n = 4) in adipocyte plasma membranes, directly demonstrating that beta3AR stimulation results in Gi-GTP exchange. The beta3AR-stimulated increase in 8-azido-[32P]GTP labeling of Galphai was equivalent to that obtained with the A1-adenosine receptor agonist N6-cyclopentyladenosine (1.56 +/- 0.07; n = 4), whereas inclusion of unlabeled GTP (100 microM) eliminated all binding. Stimulation of the beta3AR in 3T3-F442A adipocytes led to a 2-3-fold activation of mitogen-activated protein (MAP) kinase, as measured by extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation. Pretreatment of cells with pertussis toxin (PTX) eliminated MAP kinase activation by beta3AR, demonstrating that this response required receptor coupling to Gi. Expression of the human beta3AR in HEK-293 cells reconstituted the PTX-sensitive stimulation of MAP kinase, demonstrating that this phenomenon is not exclusive to adipocytes or to the rodent beta3AR. ERK1/2 activation by the beta3AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive beta3AR coupling to Gi proteins serves both to restrain Gs-mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade.     

Somatostatin receptors (sst2) are coupled to Go and modulate GTPase activity in the rabbit retina

The role of somatostatin and its mechanism of action in the retina remains an important target for investigation. Biochemical and pharmacological studies were engaged to characterize the somatostatin receptors in the rabbit retina, and their coupling to G-proteins. The ability of selective ligands to inhibit [125I]Tyr11-somatostatin-14 binding to rabbit retinal membranes was examined. The sst2 analogues SMS201-995, MK678, and BIM23014, displayed IC50 values of 0.28 +/- 0.12, 0.04 +/- 0.01 and 1.57 +/- 0.39 nm, respectively. The sst1 analogue CH275 moderately displaced the [125I]Tyr11-somatostatin-14 binding, while selective analogues for sst3, sst4 and sst5 had minimal effect. Immunoblotting and/or immunohistochemistry studies revealed the presence of the pertussis toxin sensitive Gi1/2, and Go proteins, as well as Gs. Somatostatin-14 and MK678 stimulated GTPase activity in a concentration-dependent manner with EC50 values of 42.8 +/- 16.8 and 70.0 +/- 16.5 nm, respectively, thus supporting the functional coupling between the receptor and the G-proteins. CH275 stimulated the GTPase activity moderately, in agreement with its binding profile. The antisera raised against Goalpha and Gi1/2alpha inhibited the somatostatin-induced high-affinity GTPase activity, but only anti-Goalpha inhibited the MK678 stimulation of the enzyme. These results suggest that somatostatin mediates its actions in the rabbit retina by interacting mainly with sst2 receptors that couple to Goalpha.