Guanine Nucleotide-binding Regulatory Protein Research Articles

Phosphorylation of the N-Formyl Peptide Receptor Is Required for Receptor Internalization but Not Chemotaxis

The human N-formyl peptide receptor (FPR) is a member of the family of leukocyte, G protein-coupled, chemoattractant receptors. To determine the role(s) of receptor phosphorylation in FPR processing and formylmethionylleucylphenylalanine (fMLF)-mediated chemotaxis, we utilized U937 cells expressing the recombinant wild type receptor and a mutant form of the FPR. This mutant, which lacks all of the serine and threonine residues in the C terminus of the receptor, DeltaST, has recently been shown to produce a receptor capable of fMLF binding and G protein activation but was demonstrated not to undergo fMLF-dependent phosphorylation or desensitization of the calcium mobilization response upon repeated exposure to agonist (Prossnitz, E. R. (1997) J. Biol. Chem. 272, 15213-15219). In this report, we examined the role of receptor phosphorylation in FPR internalization and leukocyte chemotaxis. Whereas the wild type receptor was rapidly internalized upon stimulation, the phosphorylation-deficient mutant was not, remaining entirely on the cell surface. In addition, contrary to the hypothesis that receptor processing and recycling are required for chemotaxis, we found no defect in the ability of the mutant FPR to migrate up a concentration gradient of fMLF. These results indicate that phosphorylation of the FPR is a necessary step in receptor internalization but that receptor phosphorylation, desensitization, and internalization are not required for chemotaxis.

Activation by β-carbolines of G-proteins in HL-60 membranes and the bovine retinal G-protein transducin in a receptor-independent manner

Naturally occurring β-carbolines are lipophilic compounds which show psychotropic and physiological effects in mammals. They bind to distinct high-affinity binding sites in various mammalian tissues. However, the mechanism by which the β-carbolines affect transmembrane signal transduction processes is still unknown. Since β-carbolines are cationic-amphiphilic substances and since such substances are known to activate heterotrimeric regulatory guanine nucleotide binding proteins (G-proteins) in a receptor-independent manner, we put forward the hypothesis that β-carbolines act directly on G-proteins. Therefore, we investigated the ability of β-carbolines to stimulate high-affinity GTP hydrolysis in membranes of dibutyryl-cAMP differentiated HL-60 cells and of the purified bovine G-protein, transducin (TD). The β-carbolines norharman and barman, stimulated the GTPase in HL-60 membranes with an ec 50 of 410 μM and 450 μM, respectively, and a maximum effect at 1 mM each. Norharman and barman stimulated the GTPase of TD with an ec 50 of 60 μM and 300 μM, and a maximum at 1 mM for both compounds. The stimulatory effect of norharman in HL-60 membranes was pertussis toxin-sensitive. Structure/activity characteristics of the β-carbolines showed a specifity of norharman to stimulate the GTPase of TD, because norharman activated GTP hydrolysis in HL-60 membranes approximately 7 times less potently than that of TD. Norharman was a five-fold more potent activator of TD than tetrahydronorharman. Hydroxylation of the β-carboline molecule in position 6 led to a loss of GTPase-activating properties. Our data suggest that naturally occurring β-carbolines are a novel class of receptor-independent G-protein activating substances. This mechanism could contribute to their diverse biological effects.

The T Cell-directed CC Chemokine TARC Is a Highly Specific Biological Ligand for CC Chemokine Receptor 4

Thymus and activation-regulated chemokine (TARC) is a recently identified CC chemokine that is expressed constitutively in thymus and transiently in stimulated peripheral blood mononuclear cells. TARC functions as a selective chemoattractant for T cells that express a class of receptors binding TARC with high affinity and specificity. To identify the receptor for TARC, we produced TARC as a fusion protein with secreted alkaline phosphatase (SEAP) and used it for specific binding. By stably transfecting five orphan receptors and five known CC chemokine receptors (CCR1 to -5) into K562 cells, we found that TARC-SEAP bound selectively to cells expressing CCR4. TARC-SEAP also bound to K562 cells stably expressing CCR4 with a high affinity (Kd = 0.5 nM). Only TARC and not five other CC chemokines (MCP-1 (monocyte chemoattractant protein-1), RANTES (regulated upon activation, normal T cells expressed and secreted), MIP-1alpha (macrophage inflammatory protein-1alpha), MIP-1beta, and LARC (liver and activation-regulated chemokine)) competed with TARC-SEAP for binding to CCR4. TARC but not RANTES or MIP-1alpha induced migration and calcium mobilization in 293/EBNA-1 cells stably expressing CCR4. K562 cells stably expressing CCR4 also responded to TARC in a calcium mobilization assay. Northern blot analysis revealed that CCR4 mRNA was expressed strongly in human T cell lines and peripheral blood T cells but not in B cells, natural killer cells, monocytes, or granulocytes. Taken together, TARC is a specific functional ligand for CCR4, and CCR4 is the specific receptor for TARC selectively expressed on T cells.

Identification of CCR6, the Specific Receptor for a Novel Lymphocyte-directed CC Chemokine LARC

Liver and activation-regulated chemokine (LARC) is a recently identified CC chemokine that is expressed mainly in the liver. LARC functions as a selective chemoattractant for lymphocytes that express a class of receptors specifically binding to LARC with high affinity. To identifiy the receptor for LARC, we examined LARC-induced calcium mobilization in cells stably expressing five CC chemokine receptors (CCR1-CCR5) and five orphan seven-transmembrane receptors. LARC specifically induced calcium flux in K562 cells as well as 293/EBNA-1 cells stably expressing an orphan receptor GPR-CY4. LARC induced migration in 293/EBNA-1 cells stably expressing GPR-CY4 with a bi-modal dose-response curve. LARC fused with secreted alkaline phosphatase (LARC-SEAP) bound specifically to Raji cells stably expressing GPR-CY4 with a Kd of 0.9 nM. Only LARC but not five other CC chemokines (MCP-1, RANTES, MIP-1alpha, MIP-1beta, and TARC) competed with LARC-SEAP for binding to GPR-CY4. By Northern blot analysis, GPR-CY4 mRNA was expressed mainly in spleen, lymph nodes, Appendix, and fetal liver among various human tissues. Among various leukocyte subsets, GPR-CY4 mRNA was detected in lymphocytes (CD4(+) and CD8(+) T cells and B cells) but not in natural killer cells, monocytes, or granulocytes. Expression of GPR-CY4 mRNA in CD4(+) and CD8(+) T cells was strongly up-regulated by IL-2. Taken together, GPR-CY4 is the specific receptor for LARC expressed selectively on lymphocytes, and LARC is a unique functional ligand for GPR-CY4. We propose GPR-CY4 to be designated as CCR6.

Role of m1 receptor-G protein coupling in cell proliferation in the prostate

The prostate gland from several animal species contains variable levels of muscarinic subtypes, but only the human prostate expresses significant levels of the m1 subtype. We studied muscarinic receptor activity in human benign prostatic hypertrophy (BPH) as well as several cell lines derived from prostate cancer. The BPH we studied expresses approximately 75% of the m1 receptor and undetectable levels of the other receptor subtypes whereas PC3 cells express only the m3 receptor subtype. DU145 and LnCaP cells express approximately equal levels of m1 and m3 receptor subtypes. Only the PC3 cells responded to carbachol with an increase in turnover of polyphosphoinositides, and none of the cell lines responded with effects on cAMP metabolism. Co-precipitation of receptors with heterotrimeric guanine nucleotide-binding regulatory proteins demonstrated interactions of the m1 receptors with Gi, Gq and G16 in BPH tissue and of the m1 and m3 receptors with Gi, Gq and G12 in PC3 and DU145 cells. Mitogen activated protein kinase (ERK) activity was seen in response to carbachol in PC3 and DU145 but not LnCaP cells. Finally, carbachol promoted cell proliferation in all three cell lines. Thus, there appears to be no consistent relationship between ERK activity, cell proliferation, and the subtype mediating the proliferative response, amongst these prostate cancer cell lines.

Muscarinic-induced mucin secretion and intracellular signaling by hamster tracheal goblet cells.

Hamster tracheal epithelial cell cultures were used to investigate muscarinic regulation of high-molecular-weight glycoconjugate (HMWG) secretion by airway goblet cells. HMWG were radiolabeled with N-acetyl-D-[1-(3)H]glucosamine, precipitated with trichloroacetic acid and phosphotungstic acid, and counted by liquid scintillation. Carbachol (100 microM) increased HMWG secretion (166.6 +/- 18.7%, P < 0.001, n = 20), and this response was blocked by the muscarinic receptor antagonist atropine. Ca2+ may not be essential for carbachol response since 1) carbachol-activated secretion was not inhibited by chelating extracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by reducing both extracellular and intracellular Ca2+ with BAPTA-acetoxymethyl ester in low-Ca2+ medium; 2) the carbachol response was only partially blocked in low-Ca2+ medium; and 3) calcium ionophore did not stimulate HMWG secretion. However, carbachol-stimulated secretion was abolished by pertussis toxin (PTX), indicating the involvement of a PTX-sensitive guanine nucleotide-binding regulatory protein (G protein), and by the protein kinase C (PKC) inhibitor chelerythrine chloride. Furthermore, carbachol-stimulated secretion was not inhibited by overnight incubation with phorbol 12-myristate 13-acetate. In conclusion, carbachol-stimulated secretion of HMWG appears to be coupled to a PTX-sensitive G protein and requires the activation of a phorbol ester-insensitive PKC isoform.

Agonist-induced high-affinity GTP hydrolysis as an index of receptor-mediated G protein activation in mammalian brain membranes.

AbstractHeterotrimeric guanine nucleotide-binding regulatory (G) proteins, composed of α-, β-, and γ-subunits, play pivotal roles in many receptor-mediated transmembrane signaling processes. Although detailed regulatory modes of G protein-mediated signal transduction have not yet been fully elucidated, the following scheme is generally accepted (Fig. 1). In an inactive state, G protein exists as an associated form of GDP-bound a-subunit and fiy-subunits (Fig. 1A). When an agonist is bound to the cell-surface receptor, the receptor is conformationally changed and interacts with the G protein so that GDP bound to the a-subunit is released and replaced with GTP (Fig. 1B). This GDP-GTP exchange reaction turns on the G protein activation process, in which GTP-bound a-subunit is dissociated from py-subunits (Fig. lC), resulting in a modulation of the function of effector molecules such as adenylyl cyclase, phospholipases, and cGMP phosphodiesterase (Fig. 1D). The a-subunit of G protein has inherent high-affinity or low-Km GTPase activity, which hydrolyzes GTP on the a-subunit into GDP and inorganic phosphate (P,) (Fig. 1E). This turn-off reaction results in reassociation of GDP-bound a-subunit and βγ-subunits to end the activation cycle of G protein (Fig. 1A). Such properties of the receptor-mediated G protein activation/inactivation cycle allow us to use several biochemical techniques to investigate the function of G proteins that are coupled with, and activated by, the receptors. These include receptormediated GDP release, GTP binding, and GTP hydrolysis. Schematic diagram of receptor-mediated activation/mactivation cycle of G protein (see Introduction for detailed comments). A, agonist; R, receptor protem; E, effector molecule; α, α-subumt of G protein; βγ, βγ-subunits of G protein; S, substrate, P, product. KeywordsActivate CharcoalBrain MembranePhosphoric Acid SolutionFinal Reaction MixturecGMP PhosphodiesteraseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Signalling properties of lysophosphatidic acid in primary human skin fibroblasts: role of pertussis toxin-sensitive GTP-binding proteins.

We have investigated the signalling properties of the naturally occurring intercellular signalling molecule lysophosphatidic acid (LPA) in primary human skin fibroblasts. LPA stimulated phospholipase C activity resulting in the formation of inositol 1,4,5-trisphosphate (IP3) which was accompanied by a concentration-dependent increase in intracellular calcium concentration ([Ca2+]i). The increase in [Ca2+]i was subject to homologous desensitisation but not to heterologous desensitisation by sphingosine-1-phosphate. The half-maximal effect of LPA on the rise in [Ca2+]i was attained at 7-20 nM. IP3 formation and Ca2+ mobilisation were highly pertussis toxin (PTX)-sensitive (100% and 75%, respectively). LPA also inhibited forskolin-stimulated formation of cAMP, which was partially reversed (51%) when fibroblasts were pretreated with PTX. To directly test the involvement of guanine nucleotide-binding regulatory proteins (G proteins), LPA-induced binding of the stable GTP analogue GTP gamma S was measured. LPA induced an increase in GTP gamma S binding, which was completely inhibited by PTX, implicating the involvement of Gi-type G proteins in LPA signalling. Furthermore, LPA increased DNA synthesis and cell proliferation. Finally, LPA induced the migration of human skin fibroblasts, which in conjunction with the stimulation of cell growth strengthens the presumed involvement of LPA in wound healing and tissue regeneration. Both effects (cell growth and migration) were almost completely PTX-sensitive. Overall, these investigations in primary cultures of human skin fibroblasts confirm and extend our knowledge about LPA signalling, suggesting a pivotal role of receptor coupled activation of Gi-type proteins at least in this cell type.

Stimulation of guanosine 5'-O-(3-[35S] thiotriphosphate) binding by cholinergic muscarinic receptors in membranes of rat olfactory bulb.

In membranes of rat olfactory bulb, a brain region in which muscarinic agonists increase cyclic AMP formation, the muscarinic stimulation of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP gamma S) binding was used as a tool to investigate the receptor interaction with the guanine nucleotide-binding regulatory proteins (G proteins). The stimulation of the radioligand binding by carbachol (CCh) was optimal (threefold increase) in the presence of micromolar concentrations of GDP and 100 mM NaCl. Exposure to N-ethylmaleimide and pertussis toxin markedly inhibited the CCh effect, whereas it increased the relative stimulation of [35S]GTP gamma S binding elicited by pituitary adenylate cyclase-activating polypeptide (PACAP). On the other hand, membrane treatment with cholera toxin curtailed the PACAP stimulation of [35S]GTP gamma S binding but did not affect the response to CCh. Like CCh, a number of cholinergic agonists stimulated [35S]GTP gamma S binding in a concentration-dependent and saturable manner. The antagonist profile of the muscarinic stimulation of [35S]GTP gamma S binding was highly correlated with that displayed by the muscarinic stimulation of adenylyl cyclase. These data indicate that the olfactory bulb muscarinic receptors couple to G1/G0, but not to Gs, and support the possibility that activation of G1/G0 mediates the stimulatory effect on adenylyl cyclase activity.

Direct evidence for two distinct G proteins coupling with thrombin receptors in human neuroblastoma SH-EP cells

Thrombin receptor-G protein coupling was investigated in the human epithelial neuroblastoma cell line, SH-EP. In these cells, both α-thrombin and thrombin receptor peptides, SFLLRNP (one-letter amino-acid code), which are newly exposed following cleavage by α-thrombin, stimulated GTPase activity about 2-fold over basal activity. Pertussis toxin treatment only partially attenuated α-thrombin-and SFLLRNP-stimulated GTPase activity by 50%, whereas antibody raised against synthetic heptapeptide SFLLRNP blocked α-thrombin-stimulated phosphoinositide hydrolysis more than 80%. Immunoprecipitation studies using this antibody showed that both G i2, a subtype of guanine nucleotide-binding regulatory proteins (G proteins) mediating inhibition of adenylyl cyclase, and G q G 11 , a G protein mediating stimulation of phospholipase C, were activated by α-thrombin. These data suggest that in these cells the thrombin receptor activates pertussis toxin-sensitive and pertussis toxin-insensitive G proteins simultaneously and directly couples to G i2 and G q G 11 , which mediate different signaling pathways.

G-protein alpha subunit Gi(alpha)2 mediates erythropoietin signal transduction in human erythroid precursors.

Erythropoietin induces a dose-dependent increase in cytosolic calcium in human erythroblasts that is mediated by a voltage-independent Ca2+ channel. Inhibition of this response to erythropoietin by pertussis toxin suggests involvement of guanine nucleotide-binding regulatory proteins (G-proteins). The role of G-proteins in regulation of the erythropoietin-modulated Ca2+ channel was delineated here by microinjection of G-protein modulators or subunits into human erythroid precursors. This is the first report on the use of microinjection to study erythropoietin signal transduction in normal precursor cells. Fura-2 loaded day-10 burst-forming units-erythroid-derived erythroblasts were used for microinjection and free intracellular calcium concentration ([Ca(i)]) was measured with digital video imaging. BCECF (1,2',7'-bis(2-carboxyethyl)-5-(and -6-)-carboxyfluorescein) was included in microinjectate, and an increase in BCECF fluorescence was evidence of successful microinjection. Cells were microinjected with nonhydrolyzable analogues of GTP, GTPgammaS or GDPbetaS, which maintain the alpha subunit in an activated or inactivated state, respectively. [Ca(i)] increased significantly in a dose-dependent manner after microinjection of GTPgammaS. However, injection of GDPbetaS blocked the erythropoietin-induced calcium increase, providing direct evidence that activation of a G-protein is required. To delineate which G-protein subunits are involved, alpha or betagamma transducin subunits were purified and microinjected as a sink for betagamma or alpha subunits in the erythroblast, respectively. Transducin betagamma, but not alpha, subunits eliminated the calcium response to erythropoietin, demonstrating the primary role of the alpha subunit. Microinjected antibodies to Gi(alpha)2, but not Gi(alpha)1 or Gi(alpha)3, blocked the erythropoietin-stimulated [Ca(i)] rise, identifying Gi(alpha)2 as the subunit involved. This was confirmed by the ability of microinjected recombinant myristoylated Gi(alpha)2, but not Gi(alpha)1 or Gi(alpha)3 subunits, to reconstitute the response of pertussis toxin-treated erythroblasts to erythropoietin. These data directly demonstrate a physiologic function of G-proteins in hematopoietic cells and show that Gi(alpha)2 is required in erythropoietin modulation of [Ca(i)] via influx through calcium channels.

Alteration of G protein subclass mRNAs in methamphetamine-induced behavioral sensitization.

Guanine nucleotide-binding regulatory proteins (G proteins) play an obligatory role in the transmembrane signaling system and are concerned with the neurochemical basis of several psychiatric disorders. We examined the alteration in the mRNA levels of G protein subclasses (Gil alpha, Gi2 alpha and Go alpha) in behavioral sensitized rats using subchronic treatment with methamphetamine (MAP). Adult male Sprague-Dawley rats received intraperitoneal injections of MAP (4 mg/kg) or saline (control) once daily for 14 days. A remarkable increase in the levels of Gi2 alpha mRNA was observed in both sides of striatum at 48 hours after the last injection of MAP. The changes in the Gi2 alpha mRNA levels were not significant at two weeks after the last injection of MAP. The levels of Go alpha and Gil alpha mRNA were not changed at 48 hours and at two weeks after the last injection of MAP. These results suggest that the changes in the Gi2 mRNA level might be responsible for the reinforcement of the generation of stereotyped abnormal behavior by repeated administration of MAP rather than the persistence of the reverse tolerance phenomenon.

The role of prenylation in G-protein assembly and function

Heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) are vital components of numerous signal transduction pathways, including sensory and hormonal response systems. G-proteins transduce signals from heptahelical transmembrane receptors to downstream effectors. The localization of a G-protein to the plasma membrane, as well as its interaction with the appropriate receptor and effector, are essential for its function. In addition, the association of a G-protein's subunits to form its trimer is required for interaction with its receptor. The G-protein γ subunits (G γ) are subject to a set of carboxyl-terminal processing events that include prenylation of a cysteine, proteolysis, and methylation. Recent advances which elucidate the contributions that the post-translational modifications of the G γ subunit have on the assembly, membrane association, and function of the G-protein trimer reveal that these modifications are required for important protein-protein, in addition to membrane-protein, interactions.

Differential changes of myocardial β-adrenoceptor subtypes and G-proteins in dogs with right-sided congestive heart failure

We have shown previously in dogs with right heart failure that the reduction of myocardial β-adrenoceptor density occurs only in the failing right ventricle, while cardiac inotropic responses to β-adrenergic stimulation are reduced in both the right and left ventricles. The purpose of the present study was to determine whether a post-receptor defect in the guanine nucleotide-binding regulatory proteins (G-proteins) existed which would explain, at least in part, the adrenergic subsensitivity in both ventricles of the heart failure dogs. Using both immunoblotting technique and the bacterial toxin-mediated ADP ribosylation assays, we found that the stimulatory G-protein (G s) was reduced in both ventricles of the heart failure dogs. In contrast, there were no changes in the inhibitory G-protein (G i). In addition, receptor subtype analysis showed that only β 1-adrenoceptors were reduced in the failing right ventricle of the heart failure animals. This study demonstrated that the reduction of β-adrenoceptors in right heart failure was chamber-specific whereas the reduction of G s was non-selective, occurring in both ventricles of right heart failure dogs. The findings further suggest that the reduction of G s probably was caused by systemic neurohormonal activation, independent of local ventricular stress.

Cytosolic Na+ controls and epithelial Na+ channel via the Go guanine nucleotide-binding regulatory protein.

In tight Na+-absorbing epithelial cells, the fate of Na+ entry through amiloride-sensitive apical membrane Na+ channels is matched to basolateral Na+ extrusion so that cell Na+ concentration and volume remain steady. Control of this process by regulation of apical Na+ channels has been attributed to changes in cytosolic Ca2+ concentration or pH, secondary to changes in cytosolic Na+ concentration, although cytosolic Cl- seems also to be involved. Using mouse mandibular gland duct cells, we now demonstrate that increasing cytosolic Na+ concentration inhibits apical Na+ channels independent of changes in cytosolic Ca2+, pH, or Cl-, and the effect is blocked by GDP-beta-S, pertussis toxin, and antibodies against the alpha-subunits of guanine nucleotide-binding regulatory proteins (Go). In contrast, the inhibitory effect of cytosolic anions is blocked by antibodies to inhibitory guanine nucleotide-binding regulatory proteins (Gi1/Gi2. It thus appears that apical Na+ channels are regulated by Go and Gi proteins, the activities of which are controlled, respectively, by cytosolic Na+ and Cl-.

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

Formation of the functional complexes of m2 muscarinic acetylcholine receptors with GTP-binding regulatory proteins in solution.

Sixteen different detergents were studied for solubilization of functional complexes between m2 muscarinic acetylcholine receptors (mAChR) and guanine nucleotide-binding regulatory proteins (G proteins). More than 40% of solubilized mAChR retained their GTP-dependent high affinity for agonist binding after solubilization with sucrose monolaurate, whereas all other detergents studied gave considerably lower solubilization yields or caused the loss of the high affinity for agonist binding. The preformation of mAChR-G protein complexes in membranes revealed that a large excess of G proteins did not increase the portion of high-affinity binding sites, but caused GTP- and Mg2(+)-dependent inhibition of the binding of radioactive antagonists to mAChR. The optimization of detergent concentration and other experimental conditions revealed that up to 47% of the solubilized receptors indicated the GTP-dependent high affinity for agonist binding after mixing solubilized mAChR with purified G proteins in sucrose monolaurate in the presence of Mg2+ and carbachol. These results give the first clear proof of the formation of functional complexes between mAChR and G proteins in solution and indicate that GTP-dependent high-affinity agonist binding is connected to the direct interactions between mAChR and G proteins and that other membrane components are not necessary.

G protein gene expression during mouse oocyte growth and maturation, and preimplantation embryo development

Fertilization in mammals initiates “egg activation,” a series of events leading to embryo development. The signal transduction events that occur as a result of sperm-egg interactions and that initiate egg activation may be analogous to a ligand-receptor-effector pathway, but the details of this signaling pathway are poorly understood. Several lines of evidence support a role for guanine nucleotide-binding regulatory proteins (G proteins) in mammalian egg activation. Prior to initiating studies to examine further the role of specific G proteins in sperm-induced mouse egg activation, we needed to define the complement of G proteins expressed in the egg. Using a reverse transcription-polymerase chain reaction (RT-PCR) assay, the relative levels of mRNAs encoding specific G protein α, β, and γ subunits were determined in meiotically incompetent oocytes, fully-grown competent oocytes, metaphase II-arrested eggs, one-, two-, and eight-cell embryos, and blastocysts. mRNA transcripts representing all of the heterotrimeric G protein families were present at all of the stages examined, and all underwent significant changes in their patterns of expression. The following heterotrimeric G protein mRNA transcripts were present in oocytes, eggs, or preimplantation embryos: Gαq family (q, 11, and 14), Gα12 family (12 and 13), Gαi family (i1, i2, i3, t2, z, and s), β subunits 1, 2, 4, and 5, and γ subunits 2, 3, 5, and 7. A recently described large molecular weight G protein, Gαh (Nakaoka et al., 1994: Science 264:1593–1596), was also present. Gα15, Gαt1, Gαolf, GαoA, Gβ3, Gγ1, and Gγ8 mRNA transcripts were not detected using this method. The most common pattern of expression observed was a maturation-associated decrease followed by an increase after the two-cell stage. Some transcripts, however, were expressed at low levels until the eight-cell to blastocyst stages, whereas others were expressed at high levels in the oocyte but following maturation declined and remained at a low level throughout preimplantation development. © 1996 Wiley-Liss, Inc.

G protein gene expression during mouse oocyte growth and maturation, and preimplantation embryo development.

Fertilization in mammals initiates "egg activation," a series of events leading to embryo development. The signal transduction events that occur as a result of sperm-egg interactions and that initiate egg activation may be analogous to a ligand-receptor-effector pathway, but the details of this signaling pathway are poorly understood. Several lines of evidence support a role for guanine nucleotide-binding regulatory proteins (G proteins) in mammalian egg activation. Prior to initiating studies to examine further the role of specific G proteins in sperm-induced mouse egg activation, we needed to define the complement of G proteins expressed in the egg. Using a reverse transcription-polymerase chain reaction (RT-PCR) assay, the relative levels of mRNAs encoding specific G protein alpha, beta, and gamma subunits were determined in meiotically incompetent oocytes, fully-grown competent oocytes, metaphase II-arrested eggs, one-, two-, and eight-cell embryos, and blastocysts. mRNA transcripts representing all of the heterotrimeric G protein families were present at all of the stages examined, and all underwent significant changes in their patterns of expression. The following heterotrimeric G protein mRNA transcripts were present in oocytes, eggs, or preimplantation embryos: G alpha q family (q, 11, and 14), G alpha 12 family (12 and 13), G alpha i family (i1, i2, i3, t2, z, and s), beta subunits 1, 2, 4, and 5, and gamma subunits 2, 3, 5, and 7. A recently described large molecular weight G protein, G alpha h (Nakaoka et al., 1994: Science 264:1593-1596), was also present, G alpha 15, G alpha t1, G alpha olf, G alpha oA, G beta 3, G gamma 1, and G gamma 8 mRNA transcripts were not detected using this method. The most common pattern of expression observed was a maturation-associated decrease followed by an increase after the two-cell stage. Some transcripts, however, were expressed at low levels until the eight-cell to blastocyst stages, whereas others were expressed at high levels in the oocyte but following maturation declined and remained at a low level throughout preimplantation development.

β-Adrenergic receptor kinase-like activity and β-arrestin are expressed in osteoblastic cells

Biologic responses to peptide calciotropic hormones, such as parathyroid hormone (PTH) and calcitonin, exhibit desensitization. As with most hormones, however, the mechanisms of desensitization are not completely understood. For the beta 2-adrenergic receptor (beta 2AR) system, which is coupled to adenylyl cyclase via the stimulatory guanine nucleotide-binding regulatory (G5) protein, homologous desensitization is mediated in part by a receptor-specific kinase (beta ARK) and a soluble cofactor (beta-arrestin). Recently, this system has been reported to be involved in rapid homologous desensitization of the PTH/parathyroid hormone receptor protein (PTHrP) receptor. We have identified the presence of this system in bone using reverse-transcriptase PCR. Nucleotide sequence of PCR fragments from ROS 17/2.8 cells revealed 100% identity with rat brain beta ARK1 and beta-arrestin 1 sequences. Northern analyses with RNA from ROS 17/2.8, UMR 106-H5 cells, and primary cultures of nontransformed neonatal rat calvariae demonstrated two mRNA species of 4 and 2.6 kilobases (kb) for beta ARK and 7.5 kb for beta-arrestin, comparable to those found in bovine brain. beta ARK-like activity was demonstrated in cytosolic extracts of the UMR 106-H5 cells by assessing phosphorylation of the retinal photoreceptor, rhodopsin, by the extracts. Phosphorylation was enhanced with light-activated rhodopsin and by bovine brain G beta gamma subunits; heparin inhibited phosphorylation. These findings are characteristic of beta ARK. Expression of beta-arrestin in the UMR 106-H5 cells was confirmed by immunoblot. Thus, osteoblastic cells express proteins, beta ARK, and beta-arrestin, which may regulate desensitization of calciotropic hormone receptors.