Pregnancy-related modifications of rat myometrial Gs proteins: ADP ribosylation, immunoreactivity and gene expression studies

Previous studies from our laboratory have suggested that post-receptor events at the level of beta-adrenergic receptor-adenylate cyclase interaction could be altered in myometrium by steroid hormones or pregnancy. In this study, we have addressed this question by performing a direct evaluation of rat myometrial Gs proteins at various stages of pregnancy or 24 h after administration of progesterone. In the 50,000 g myometrial plasma membrane fraction, in the presence of 32P-labelled NAD, cholera toxin ribosylated three predominant proteins with apparent molecular masses of 42, 47 and 55 kDa. Western blot analysis using the RM/1 antibody recognized the 42 and 47 kDa cholera toxin ADP-ribosylated bands but not the 55 kDa band. Thus, the 42 and 47 kDa immunoreactive bands were interpreted as being the small (Gs alpha-S) and large (Gs alpha-L) forms of Gs respectively. With a more purified myometrial plasma membrane fraction (105,000 g) an additional minor band of 44 kDa could be observed with both techniques. Treatment of late pregnant rats with 5 mg progesterone resulted in a significant increase in both Gs alpha subunits: +25% and +30% after ADP-ribosylation, +50% and +60% after Western blot analysis for Gs alpha-L and Gs alpha-S respectively. Pretreatment with the antiprogestin RU 486 completely suppressed the effect of progesterone, suggesting that the expression of Gs alpha subunits may be under the control of progesterone. However, changes in the myometrial content of Gs in progesterone-treated rats were not associated with concomitant variations in the steady-state levels of mRNA as demonstrated by Northern blot analysis. These data suggest a post-translational regulation of Gs expression by progesterone. Amounts of ADP-ribosylated Gs showed characteristic changes during the course of pregnancy with a fourfold or threefold increase (P < 0.05) on day 15 versus day 12 or delivery respectively. During pregnancy, or after progesterone administration, myometrial alterations of Gs strongly correlated (r = 0.913, P < 0.01) with the cholera toxin-stimulated adenylate cyclase activity. These findings provide evidence that changes in myometrial amounts of functional Gs i) are controlled by the hormonal status of pregnancy and progesterone and ii) play an important role in the transduction pattern of adenylate cyclase activity during the course of pregnancy.     

G proteins in adipocytes and preadipocytes: characterization, subcellular distribution, and potential roles for Gi2 and/or Gi3 in the control of cell proliferation

Guanosine triphosphate (GTP)-binding protein subunits were studied by immunoblot analysis in particulate fractions from mature adipocytes, confluent preadipocytes, and in vitro-differentiated preadipocytes. Mature adipocytes express Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha, Gq/11 alpha, G13 alpha and the long and short isoforms of Gs alpha, but no Gz alpha or G12 alpha. Confluent and differentiated preadipocytes differ in having a higher content of Gi alpha 3 and G13 alpha and expressing G12 alpha. In contrast, they lack Gi alpha 1, Go alpha, and the short from of Gs alpha. The G-protein alpha subunits Gi alpha 2, Gs alpha (long isoform), and Gq/11 alpha, and G-protein beta subunits were unchanged throughout the differentiation process. By immunoblot and indirect immunofluorescence studies on confluent preadipocytes, we showed that Gi alpha 2 is present in the endoplasmic reticulum and marginally in plasma membranes and nuclei. In contrast, antibodies to Gi alpha 3 stained the Golgi apparatus. The role of G proteins on preadipocyte proliferation was studied using Bordetella pertussis toxin. Exposure of growing cells to this toxin in the presence of fetal calf serum (FCS) decreased [3H]thymidine incorporation by 40% and induced a 40% increase in doubling time. This resulted in a 30% decrease in cell number per well after 48 h. These effects of B. pertussis toxin did not appear to be related to an increase in cyclic adenosine monophosphate (cAMP) concentration, because forskolin had the opposite effect on cell proliferation. Finally, B. pertussis toxin prevented serum-induced Raf1 association to the plasma membrane, possibly by disrupting FCS-induced G beta gamma effects on the Ras/Raf1 pathway. Since Go alpha and Gi alpha 1 subunits were absent in preadipocytes, we conclude that Gi2 and/or Gi3 proteins transduce some mitogenic signals of FCS through release of G beta gamma subunits. The subcellular distribution of Gi alpha 2 and Gi alpha 3 suggests that part of their functions result from interactions with components other than the plasma membrane.     

Differential activity of the G protein beta5 gamma2 subunit at receptors and effectors

The G protein beta5 subunit differs substantially in amino acid sequence from the other known beta subunits suggesting that beta gamma dimers containing this protein may play specialized roles in cell signaling. To examine the functional properties of the beta5 subunit, recombinant beta5 gamma2 dimers were purified from baculovirus-infected Sf9 insect cells using a strategy based on two affinity tags (hexahistidine and FLAG) engineered into the N terminus of the gamma2 subunit (gamma2HF). The function of the pure beta5 gamma2HF dimers was examined in three assays: activation of pure phospholipase C-beta in lipid vesicles; activation of recombinant, type II adenylyl cyclase expressed in Sf9 cell membranes; and coupling of alpha subunits to the endothelin B (ETB) and M1 muscarinic receptors. In each case, the efficacy of the beta5 gamma2HF dimer was compared with that of the beta1 gamma2HF dimer, which has demonstrated activity in these assays. The beta5 gamma2HF dimer activated phospholipase C-beta with a potency and efficacy similar to that of beta1 gamma2 or beta1 gamma2HF; however, it was markedly less effective than the beta1 gamma2HF or beta1 gamma2 dimer in its ability to activate type II adenylyl cyclase (EC50 of approximately 700 nM versus 25 nM). Both the beta5 gamma2HF and the beta1 gamma2HF dimers supported coupling of M1 muscarinic receptors to the Gq alpha subunit. The ETB receptor coupled effectively to both the Gi and Gq alpha subunits in the presence of the beta1 gamma2HF dimer. In contrast, the beta5 gamma2HF dimer only supported coupling of the Gq alpha subunits to the ETB receptor and did not support coupling of the Gi alpha subunit. These results suggest that the beta5 gamma2HF dimer binds selectively to Gq alpha subunits and does not activate the same set of effectors as dimers containing the beta1 subunit. Overall, the data support a specialized role for the beta5 subunit in cell signaling.     

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.     

In vivo injection of antisense oligodeoxynucleotides to G alpha subunits and supraspinal analgesia evoked by mu and delta opioid agonists

For 5 consecutive days repeated intracerebroventricular (i.c.v.) administration of antisense oligodeoxynucleotides (ODNs) to G alpha subunit mRNAs was used to impair the function of mouse Gi1, Gi2, Gi3 and Gx/z regulatory proteins. Decreases of 20 to 60% on the G alpha-like immunoreactivity could be observed in neural structures of mouse brain, an effect that was not produced by a random-sequence ODN used as a control. The ODN to Gi1 alpha subunits lacked effect on opioid-evoked analgesia. In mice injected with the ODN to Gi2 alpha subunits the antinociceptive activity of all the opioids studied appeared greatly impaired. The ODN to Gi3 alpha subunits reduced the effects of the selective agonists of delta opioid receptors, [D-Pen2,5]-enkephalin and [D-Ala2]deltorphin II. Conversely, the analgesia evoked by opioids binding mu opioid receptors, [D-Ala2, N-MePhe4,Gly-ol5]enkephalin and morphine, appeared consistently and significantly attenuated in mice injected with the ODN to Gx/z alpha. The effect of the neuropeptide beta-endorphine-(1-31) agonist at mu and delta receptors was also reduced by ODNs to Gi3 alpha or Gx/z alpha subunits. l.c.v. injection of antibodies directed to these G alpha subunits antagonized opioid-induced analgesia with a pattern similar to that observed for the ODNs. Thus, the mu and delta opiod receptors regulate different classes of G transducer proteins to mediate the analgesic effect of agonists. The in vivo antisense strategy and the use of specific antibodies to G alpha subunits gave comparable results, indicating that in the neural tissue the mRNAs and the G alpha subunits can be accessed by the corresponding ODNs and IgGs.     

Abnormal G protein alpha(s) - and alpha(i2)-subunit mRNA expression in bipolar affective disorder

Disturbances of events associated with intracellular signaling pathways have been suspected of involvement in the development or progression of affective disorders. Often, heterotrimeric G proteins are located at the beginning of these pathways as modulators of extracellular messages. For this reason, messenger RNA expression of three G protein alpha-subunits and of phosphatidylinositol-3 kinase (PI-3 K) regulatory subunit p85 was examined in granulocytes from patients with bipolar or unipolar affective disorder and compared to healthy controls. Messenger RNA expression of the G protein subunit alpha(q) and of p85 was identical in unipolar and bipolar patients and in controls. Furthermore, mRNAs of G protein subunits alpha(s) and alpha(i2) were not different in unipolar patients as compared to healthy controls. Alpha(s) mRNA, however, was markedly increased in bipolar patients. This increase was observed in lithium-treated (more than 12 months) and in unmedicated patients. Elevated levels of alpha(i2) mRNA in unmedicated bipolar patients did not reach statistical significance, whereas mRNA in bipolar patients receiving lithium was significantly above controls. Finally, long-term medication of unipolar patients with lithium had no influence on alpha(i2) mRNA levels. The data reveal elevated mRNA levels of G alpha(s) as a robust feature of bipolar affective disorder. Moreover, despite responsiveness of alpha(i2) gene expression to cAMP-related events, no substantial upregulation of alpha(i2) mRNA was observed in bipolar patients. The lack of alpha(i2) mRNA upregulation, hence, could be an additional abnormality in these patients. Even though lithium was able to reinstate this upregulation, there was no feedback downregulation of alpha(s). This strongly supports the notion of major disturbances of the cAMP signaling system in bipolar illness.     

RGS proteins determine signaling specificity of Gq-coupled receptors

Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits, thereby attenuating signaling. RGS4 is a GTPase-activating protein for Gi and Gq class alpha subunits. In the present study, we used knockouts of Gq class genes in mice to evaluate the potency and selectivity of RGS4 in modulating Ca2+ signaling transduced by different Gq-coupled receptors. RGS4 inhibited phospholipase C activity and Ca2+ signaling in a receptor-selective manner in both permeabilized cells and cells dialyzed with RGS4 through a patch pipette. Receptor-dependent inhibition of Ca2+ signaling by RGS4 was observed in acini prepared from the rat and mouse pancreas. The response of mouse pancreatic acini to carbachol was about 4- and 33-fold more sensitive to RGS4 than that of bombesin and cholecystokinin (CCK), respectively. RGS1 and RGS16 were also potent inhibitors of Gq-dependent Ca2+ signaling and acted in a receptor-selective manner. RGS1 showed approximately 1000-fold higher potency in inhibiting carbachol than CCK-dependent signaling. RGS16 was as effective as RGS1 in inhibiting carbachol-dependent signaling but only partially inhibited the response to CCK. By contrast, RGS2 inhibited the response to carbachol and CCK with equal potency. The same pattern of receptor-selective inhibition by RGS4 was observed in acinar cells from wild type and several single and double Gq class knockout mice. Thus, these receptors appear to couple Gq class alpha subunit isotypes equally. Difference in receptor selectivity of RGS proteins action indicates that regulatory specificity is conferred by interaction of RGS proteins with receptor complexes.     

Functional coupling of NKR-P1 receptors to various heterotrimeric G proteins in rat interleukin-2-activated natural killer cells

NKR-P1 molecules constitute a family of type II membrane receptors in natural killer (NK) cells that preferentially activate NK cell killing and release of interferon-gamma from these cells. Here, we demonstrate that anti-NKR-P1 enhances GTP binding in rat interleukin-2-activated NK cell membranes; GTP binding to Gi3alpha, Gsalpha, Gq,11alpha, and Gzalpha increased noticeably in these cell membranes after treatment with anti-NKR-P1. Western blot analysis of membrane proteins prepared from interleukin-2-activated NK cells reveals the presence of Gi1,2alpha, Gi3alpha, Goalpha, Gsalpha, Gq, 11alpha, Gzalpha, and G12alpha, but not G13alpha. However, only alphai3, alphas, alphaq,11, and alphaz, but not alphai1,2, alphao, alpha12, or alpha13 subunits when immunoprecipitated with the appropriate anti-G protein antibodies, are associated with NKR-P1 when immunoblotted with anti-NKR-P1. Reciprocally, NKR-P1 immunoprecipitated with anti-NKR-P1 is associated with alphai3, alphas, alphaq,11, and alphaz immunoblotted with anti-G proteins. These results are the first to demonstrate the physical and functional coupling of NKR-P1 to the heterotrimeric G proteins in NK cells.     

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.     

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.     

Transformation of Rat-1 fibroblasts with the v-src oncogene increases the tyrosine phosphorylation state and activity of the alpha subunit of Gq/G11

Two major intermediaries in signal transduction pathways are pp60v-sre family tyrosine kinases and heterotrimeric guanine nucleotide-binding proteins. In Rat-1 fibroblasts transformed by the v-src oncogene, endothelin-1 (ET-1)-induced inositol 1,4,5-trisphosphate accumulation is increased 6-fold, without any increases in the numbers of ET-1 receptors or in the response to another agonist, thrombin. This ET-1 hyperresponse can be inhibited by an antibody directed against the carboxyl terminus of the Gq/G11 alpha subunit, suggesting that the Gq/G11 protein couples ET-1 receptors to phospholipase C (PLC). While v-src transformation did not increase the expression of the Gq/G11 alpha subunit, immunoblotting with anti-phosphotyrosine antibodies and phosphoamino acid analysis demonstrated that the Gq/G11 alpha subunit becomes phosphorylated on tyrosine residues in v-src-transformed cells. Moreover, when the Gq/G11 protein was extracted from control and transformed cell lines and reconstituted with exogenous PLC, AIF*4-stimulated Gq/G11 activity was markedly increased in extracts from v-src-transformed cells. Our results demonstrate that the process of v-src transformation can increase the tyrosine phosphorylation state of the Gq/G11 alpha-subunit in intact cells and that the process causes an increase in the Gq/G11 alpha-subunit's ability to stimulate PLC following activation with AIF-4.     

The disaggregation theory of signal transduction revisited: further evidence that G proteins are multimeric and disaggregate to monomers when activated

We have compared the sedimentation rates on sucrose gradients of the heterotrimeric GTP-binding regulatory (G) proteins Gs, G(o), Gi, and Gq extracted from rat brain synaptoneurosomes with Lubrol and digitonin. The individual alpha and beta subunits were monitored with specific antisera. In all cases, both subunits cosedimented, indicating that the subunits are likely complexed as heterotrimers. When extracted with Lubrol all of the G proteins sedimented with rates of about 4.5 S (consistent with heterotrimers) whereas digitonin extracted 60% of the G proteins with peaks at 11 S; 40% pelleted as larger structures. Digitonin-extracted Gi was cross-linked by p-phenylenedimaleimide, yielding structures too large to enter polyacrylamide gels. No cross-linking of Lubrol-extracted Gi occurred. Treatment of the membranes with guanosine 5'-[gamma-thio]triphosphate and Mg2+ yielded digitonin-extracted structures with peak sedimentation values of 8.5 S--i.e., comparable to that of purified G(o) in digitonin and considerably larger than the Lubrol-extracted 2S structures representing the separated alpha and beta gamma subunits formed by the actions of guanosine 5'-[gamma-thio]triphosphate. It is concluded that the multimeric structures of G proteins in brain membranes are at least partially preserved in digitonin and that activation of these structures in membranes yields monomers of G proteins rather than the disaggregated products (alpha and beta gamma complexes) observed in Lubrol. It is proposed that hormones and GTP affect the dynamic interplay between multimeric G proteins and receptors in a fashion analogous to the actions of ATP on the dynamic interactions between myosin and actin filaments. Signal transduction is mediated by activated monomers released from the multimers during the activation process.     

Prostaglandin G/H synthase (PGHS)-2 expression in bovine myometrium: influence of steroid hormones and PGHS inhibitors

Prostaglandins (PGs) are important mediators regulating uterine functions during the reproductive process. The objective of this study was to examine, in myocytes from the circular and longitudinal layers of bovine myometrium, the relative levels of mRNA and proteins corresponding to the gene expression of key enzymes (phospholipase A2; prostaglandin G/H synthase-1 [PGHS-1]; prostaglandin G/H synthase-2 [PGHS-2]; prostaglandin I2 synthase) involved in PG biosynthesis. We examined the influence of estradiol-17beta and progesterone on the expression and activity of these enzymes. Treatment of myocytes with progesterone (P4: 10 nM, 24 h) in the absence or presence of estradiol-17beta (E2: 1 nM, 72 h) suppressed PG biosynthesis by approximately 60% in both myometrial layers. No significant effect was observed after E2 treatment. The combined effect of E2 and P4 on PG accumulation was correlated with the modulation of PGHS-2 protein and mRNA levels in the two myometrial layers without affecting other enzymes of the PG cascade. Selective or nonselective inhibition of PGHS activity with CGP 28238 (PGHS-2-specific; a product from Ciba-Geigy: 6-[2, 4-difluorophenoxy]-5-methyl-sulfonylamino-1-indanone) or indomethacin (PGHS-1 and -2) reduced prostacyclin accumulation (measured as 6-keto-PGF1alpha in the culture medium) in a dose-dependent manner in the two myometrial layers. A significant inhibitory effect was obtained at a low concentration of indomethacin (1 nM, p < 0.05) compared to CGP 28238 (10 nM, p < 0. 05). In both myometrial layers, the maximal effect of indomethacin and/or CGP 28238 on PG accumulation was observed at 100 nM and represented 85% and 65% inhibition, respectively. In the presence of phorbol 12-myristate (100 nM), CGP 28238 (10 nM) significantly suppressed PGHS-2 mRNA level by 44.80 +/- 7.67% (p < 0.01) and 27.83 +/- 7.62% (p < 0.05) in the longitudinal and circular layer, respectively. In contrast, indomethacin did not have any significant effect. These data constitute the first quantitative analysis of key enzymes involved in PG biosynthesis in separated myometrial layers. Furthermore, the results provide interesting information on the CGP 28238 drug modulating both enzymatic activity and mRNA expression of PGHS-2.     

delta Opioid receptor subtypes activate inositol-signaling pathways in the production of antinociception

To analyze the selectivity of delta receptor subtypes to regulate different classes of G proteins, the expression of the alpha-subunits of Gi2, Gi3, Go1, Go2, Gq and G11 transducer proteins was reduced by administration of oligodeoxynucleotides (ODNs) complementary to sequences in their respective mRNAs. Mice receiving antisense ODNs to Gi2 alpha, Gi3 alpha, Go2 alpha and G11 alpha subunits showed an impaired antinociceptive response to all the delta agonists evaluated. An ODN to Go1 alpha specifically blocked the antinociceptive effect of the agonist of delta-1 receptors, [D-Pen2,5]enkephalin (DPDPE), without altering the activity of [D-Ala2]deltorphin II or [D-Ser2]-Leu-enkephalin-Thr (DSLET). In mice treated with an ODN to Gq alpha, the effects of the agonists of delta-2-opioid receptors were reduced, but not those of DPDPE. Thus, Go1 proteins are selectively linked to delta-1-mediated analgesia, and Gq proteins are related to delta-2-evoked antinociception. After impairing the synthesis of Go1 alpha subunits, DPDPE exhibited an antagonistic activity on the antinociception produced by [D-Ala2]deltorphin II. After treatment with ODNs complementary to sequences in Gq alpha or PLC-beta 1 mRNAs, the analgesic capacity of [D-Ala2]deltorphin II was diminished. However, the delta-2-agonist did not alter the antinociceptive activity of DPDPE. An ODN complementary to nucleotides 7 to 26 of the murine delta receptor reduced the analgesic potency of [D-Ala2]deltorphin II, but not that observed for DPDPE. In these mice, [D-Ala2]deltorphin II did not antagonize the effect of DPDPE. These results suggest the existence of different molecular forms of the delta opioid receptor, and the involvement of inositol-signaling pathways in the supraspinal antinociceptive effects of delta agonists.     

A single mutation Asp229 --> Ser confers upon Gs alpha the ability to interact with regulators of G protein signaling

RGS proteins (regulators of G protein signaling) are GTPase activating proteins (GAPs) for Gi and Gq families of heterotrimeric G proteins but have not been found to interact with Gs alpha. The Gs alpha residue Asp229 has been suggested to be responsible for the inability of RGS proteins to interact with Gs alpha [Natochin, M., and Artemyev, N. O. (1998) J. Biol. Chem. 273, 4300-4303]. To test this hypothesis, we have investigated the possibility of generating an interaction between Gs alpha and RGS proteins by substituting Gs alpha Asp229 with Ser and replacing the potential Gs alpha Asp229 contact residues in RGS16, Glu129 and Asn131, by Ala and Ser, respectively. RGS16 and its mutants failed to interact with Gs alpha. A single mutation of Gs alpha, Asp229Ser, rendered the Gs alpha subunit with the ability to interact with RGS16 and RGS4. Like RGS protein binding to Gi and Gq alpha-subunits, RGS16 preferentially recognized the AlF4--bound conformation of Gs alpha Asp229Ser. In a single-turnover assay, RGS16 maximally stimulated GTPase activity of Gs alpha Asp229Ser by approximately 5-fold with an EC50 value of 7.5 microM. Our findings demonstrate that Asp229 of Gs alpha represents a major barrier for Gs alpha interaction with known RGS proteins.     

Localization and sex steroid regulation of androgen receptor gene expression in rhesus monkey uterus

OBJECTIVE: To characterize the cellular sites and hormonal regulation of uterine androgen receptor gene expression in the monkey. METHODS: Ovariectomized rhesus monkeys (five in each group) were treated with placebo (the control group), estradiol (E2), E2 plus progesterone, or E2 plus testosterone by sustained-release pellets administered subcutaneously. After 3 days of treatment, uteri were removed and uterine sections were analyzed by in situ hybridization for androgen receptor messenger RNA (mRNA). RESULTS: Androgen receptor mRNA was detected in endometrial stromal cells and myometrial smooth muscle cells, with lesser expression in endometrial epithelial cells. Both E2 and E2 plus progesterone treatment doubled androgen receptor mRNA levels in stromal cells (P < .01), whereas E2 plus testosterone treatment increased stromal androgen receptor mRNA levels by about five-fold (P < .001) compared with placebo treatment. In the endometrial epithelium, E2 alone did not increase androgen receptor mRNA levels significantly. However, the E2 plus progesterone and E2 plus testosterone treatments increased epithelial androgen receptor mRNA levels by 4.3 and 5 times, respectively (P = .008 and P < .002, respectively). Androgen receptor mRNA was distributed homogeneously in smooth muscle cells across the myometrium. Estradiol treatment alone did not increase myometrial androgen receptor mRNA levels significantly, but the E2 plus progesterone and E2 plus testosterone treatments increased myometrial androgen receptor mRNA levels by 1.8 and 2 times, respectively (P = .001 and P < .001, respectively). CONCLUSION: Androgen receptor gene expression was detected in all uterine cell compartments where it was subject to significant sex steroid regulation. The fact that androgen receptor mRNA levels were consistently up-regulated by a combined E2 plus testosterone treatment while E2 treatment alone had little or no effect shows that a collaborative action of E2 and testosterone enhances androgen receptor expression in the monkey uterus.     

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.     

Localization of a G protein Gi2 in the cilia of rat ependyma, oviduct and trachea

In previous studies, the localization of a pertussis toxin-sensitive G protein was demonstrated in ependymal cilia, but the identification of the subtype of G protein was inconsistent. To clarify this issue, we studied the localization of Goalpha, Gi1alpha, Gi3alpha and Gi2alpha in the ciliated ependymal cells and in the cilia of some other tissues of rats using specific antibodies. The cilia of the ependymal cells that line the ventricular cavity of the brain were intensely immunoreactive for Gi2alpha, but not for Goalpha, Gi1alpha or Gi3alpha. Immunoblot analysis demonstrated higher levels of Gi2alpha in the ependymal cilia-rich pellet than in the motor area of the parietal cortex. At the ultrastructural level, the immunoreactivity specific for Gi2alpha was found predominantly in the cilia, but rarely in the microvilli or the basal bodies of ependymal cells. In cross-sections, the immunoreactivity specific for Gi2alpha was observed only in cell membranes, in particular, in the inner electron-dense leaflet of the trilaminar structure. In addition to that in the ependymal cilia, such specific localization of Gi2alpha was observed in the motile cilia in other tissues, including the oviduct and trachea. By contrast, the stereocilia in the ductus deferens were not immunopositive for Gi2alpha. These findings suggest that Gi2 might play an important role in the signal transduction in ciliary membrane-associated function(s) of the ependymal cells, oviduct and trachea.