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H Kurose

Publications and source records attributed to H Kurose.

At least 37 records · Page 2Linked to original sources
PubMed1998

[Production of various antibodies and single chain Fv molecule against signal-transducing proteins].

In the present review, we described the procedures of production of polyclonal and monoclonal antibodies and single chain Fv molecule (scFv). Among several animal species, the rabbit is the best animal for polyclonal antibody production and the mouse is the best animal for monoclonal antibody production. In this review, we discussed problems that might be encountered when trying to produce antibodies. Polyclonal antibodies are easily produced in rabbits by immunizing them with glutathione-S-transferase fusion proteins. However, it is difficult to eliminate nonspecifically reacting antibodies, even after the antibodies were purified from sera by an antigen column. Monoclonal antibody production is a time-consuming process, but it successful, will produce a very useful reagent due to no limitation of supply and constant quality. We described monoclonal antibody production by means of glutathione-S-transferase fusion protein. scFv is a portion of the antibody and is constructed by PCR of VH and VL regions of the antibody. We recommend that scFv should be constructed from a hybridoma that secretes monoclonal antibody, although some researchers have claimed that scFv can be constructed from the spleen of immunized mice. The expression of scFv is a promising approach to analyze the function of one of the subtypes, when the original monoclonal antibody can block the function of the protein.

Animals↗
PubMed1998

[Functional analysis of G protein-coupled receptor kinase (beta ARK1) by intracellular immunization].

G protein-coupled receptor kinases (GRKs) are believed to involve in desensitization of the G protein-coupled receptors. So far, cDNAs of six GRKs were cloned from several species including human and rat. However, it is unknown whether single GRK phosphorylates various receptors and desensitizes them in the cells. To determine whether GRK2 (also called beta ARK1) involves desensitization of the beta 1-adrenergic receptor-mediated response in heart, we tried to apply monoclonal antibody which could recognize only beta ARK1 and inhibit its phosphorylating activity to the heart cells. Monoclonal antibody was obtained by immunization of carboxyl terminus of beta ARK1 as fusion protein of glutathione-S-transferase (GST). The resulting monoclonal antibody specifically reacted with beta ARK1, and inhibited the binding of purified beta gamma subunit to the carboxyl terminus. Monoclonal antibody completely inhibited phosphorylation of the m2 muscarinic acetylcholine receptor as well as phosphorylation of GST-intracellular third loop fusion protein of the m2 receptor. When monoclonal antibody was applied to myocyte prepared from guinea pig heart, the desensitization of the beta 1-adrenergic receptor was partially inhibited as measured by Ca2+ channel activation. Thus intracellular application of monoclonal antibody is promising approach to analyze function of GRKs.

Animals↗
PubMed1998

Identification of a key amino acid of the beta2-adrenergic receptor for high affinity binding of salmeterol.

Transmembrane domains (TMDs) I, II, and VII of the beta2-adrenergic receptor (beta2AR) were replaced, individually or in combination, with the corresponding regions of the beta1AR, and vice versa. The beta2-selective binding of salmeterol was not affected by the exchange of TMD I between the beta1- and beta2ARs. The affinity of salmeterol was slightly decreased (32-fold) by replacement of TMD II of the beta2AR with the homologous region of the beta1AR; the affinity was strongly decreased (1870-fold) for the beta2AR with TMD VII of the beta1AR. The affinity of salmeterol was partially restored by the introduction of TMD VII, but not TMD II, of the beta2AR into the beta1AR. By analyzing alanine-substituted mutants, we found that Tyr308 in TMD VII was mainly responsible for the high affinity binding of salmeterol. Two salmeterol derivatives with the ether oxygen at different positions in the side chain showed 33- and 64-fold decreased affinities for the wild-type beta2AR, and a derivative with no ether oxygen showed 147-fold decreased affinity for the wild-type beta2AR. These results indicate that Tyr308 in TMD VII is the major amino acid conferring the beta2-selective binding of salmeterol to the beta2AR and that the position of the ether oxygen in the side chain is also important for beta2-selective binding. A three-dimensional model of the salmeterol-beta2AR complex shows that the phenyl group of Tyr308 interacts with methylene groups near the protonated amine of salmeterol and the ether oxygen interacts with Tyr316.

Adrenergic beta-Agonists↗
PubMed1997

Differential contribution of two serine residues of wild type and constitutively active beta2-adrenoceptors to the interaction with beta2-selective agonists.

1. We have studied the difference in receptor binding activity between partial and full beta2-adrenoceptor agonists and the abilities of the agonists to interact with Ser204 and Ser207 in the fifth transmembrane region of the beta2-adrenoceptor, amino acid residues that are important for activation of the beta2-adrenoceptor. 2. In the binding study with [125I]-iodocyanopindolol, the Ki values of (+/-)-salbutamol, (+/-)-salmeterol, TA-2005 and (-)-isoprenaline for the beta2-adrenoceptor expressed in COS-7 cell membranes were 3340, 21.0, 12.0 and 904 nM, respectively. The beta1/beta2 selectivity of these agonists was in the order of (+/-)-salmeterol (332 fold) > TA-2005 (52.8) > (+/-)-salbutamol (6.8) > (-)-isoprenaline (1.1), and the beta3-/beta2-adrenoceptor selectivity of these agonists was in the order of TA-2005 (150 fold) > (+/-)-salmeterol (88.6) > (+/-)-salbutamol (10.4) > (-)-isoprenaline (3.2). 3. The maximal activation of adenylyl cyclase by stimulation of the beta1-, beta2- and beta3-adrenoceptors by TA-2005 was 32, 100 and 100% of that by (-)-isoprenaline, respectively, indicating that TA-2005 is a full agonist at the beta2- and beta3-adrenoceptors and a partial agonist at the beta1-adrenoceptor. (+/-)-Salbutamol and (+/-)-salmeterol were partial agonists at both beta1- (8% and 9% of (-)-isoprenaline) and beta2- (83% and 74% of (-)-isoprenaline) adrenoceptors. 4. The affinities of full agonists, TA-2005 and (-)-isoprenaline, were markedly decreased by substitution of Ala for Ser204 (S204A) of the beta2-adrenoceptor, whereas this substitution slightly reduced the affinities of partial agonists, (+/-)-salbutamol and (+/-)-salmeterol. Although the affinities of full agonists for the S207A-beta2-adrenoceptor were decreased, those of partial agonists for the S207A-beta2-adrenoceptor were essentially the same as for the wild type receptor. 5. The constitutively active mutant (L266S, L272A) of the beta2-adrenoceptor had an increased affinity for all four agonists. The affinities of full agonists were decreased by substitution of Ser204 of the constitutively active mutant, whereas the degree of decrease was smaller than that caused by the substitution of the wild type receptor. Although the affinities of (+/-)-salbutamol and (+/-)-salmeterol for the S207A-beta2-adrenoceptor were essentially the same as those for the wild type beta2-adrenoceptor, the affinities of (+/-)-salbutamol and (+/-)-salmeterol for the constitutively active beta2-adrenoceptor were decreased by substitution of Ser207. 6. These results suggest that Ser204 and Ser207 of the wild type and constitutively active beta2-adrenoceptors differentially interacted with beta2-selective agonists.

Adenylyl Cyclases↗
PubMed1997

Relationship between desensitization and downregulation of beta-adrenoceptors in cardiac tissues after prolonged in vivo infusion of T-0509, a beta 1-adrenoceptor agonist.

To examine the contribution of beta-adrenoceptor (beta AR) downregulation to desensitization of beta ARs by chronic administration of a beta AR agonist, we compared the adenylyl cyclase (AC) activities in two kinds of cardiac ventricular membranes with decreased available beta ARs: one was derived from rats infused with a selective beta 1 AR agonist, T-0509 [(-)-(R)-1-(3,4-dihydroxyphenyl)- 2-[(3,4-dimethoxyphenethyl)-amino]ethanol hydrochloride], in vivo (40 micrograms/kg/hr, s.c. for 6 days); and the other was obtained from treatment of control membranes with an irreversible beta AR antagonist, bromoacetyl alprenolol methane (BAAM). T-0509 infusion decreased the densities of beta 1 ARs and beta 2 ARs by 26% and 32%, respectively, and reduced the maximal isoproterenol-stimulated AC activity by 53%. The amount of Gs alpha and Gi alpha proteins in the membranes was not significantly changed by T-0509 infusion. To make preparations that mimic the T-0509-induced downregulation, we treated the control membranes with 100 nM BAAM in vitro. The BAAM treatment decreased the Bmax value of [125I]iodocyanopindolol for beta 1 ARs and beta 2 ARs by 29% and 36%, respectively, whereas it reduced the maximal effect of isoproterenol on AC activity only by 37%. These results suggest that downregulation of beta ARs cannot fully account for the desensitization by chronic treatment of T-0509 and that other mechanism(s) can play a significant role in the loss of responsiveness.

Adenylyl Cyclases↗
PubMed1996

Molecular characterization of pharmacological properties of T-0509 for beta-adrenoceptors.

The pharmacological properties of T-0509, (-)-(R)-1-(3,4-dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol, were compared with those of isoproterenol. In the radioligand binding studies of [125I]iodocyanopindolol with COS-7 cell membranes that transiently expressed beta-adrenoceptor subtypes, T-0509 exhibited 11- and 97-fold greater Ki values for beta 2- and beta 3-adrenoceptors, respectively, compared with beta 1-adrenoceptors. Affinities of beta 2- and beta 3-adrenoceptors to isoproterenol were 1.4- and 28-fold lower than that of beta 1-adrenoceptors, respectively. The maximal stimulatory effects of T-0509 on adenylyl cyclase of CHO-K1 (chinese hamster ovary K1) cell membranes expressing beta 1- or beta 2-adrenoceptors were 85% or 96% of those produced by isoproterenol, respectively. These results indicate that T-0509 is a relatively specific beta 1-adrenoceptor agonist with a high intrinsic activity as compared with isoproterenol.

Adenylyl Cyclases↗
PubMed1996

Cellular and subcellular distribution of alpha 2A-adrenergic receptors in the visual cortex of neonatal and adult rats.

Activation of alpha 2-adrenergic receptors (alpha 2AR) in the cerebral cortex has been shown to modulate visually guided delayed response tasks as well as anxiety and depression. We used an antiserum directed specifically against the A subtype of alpha 2AR (alpha 2AAR) to determine the cell types and subcellular sites for noradrenergic reception mediated by this receptor in the adult and the developing rat visual cortices. Light microscopic examination of adult tissue revealed numerous labeled perikarya in layers II-VI, many of which appeared distinctly pyramidal. A few perikarya in layer I also were immunoreactive. In all layers, alpha 2AAR immunoreactivity (alpha 2AAR-ir) was present within proximal dendrites and fine processes. In neonatal tissue, there was an intense, distinct band of immunoreactivity spanning the layer composed of tightly packed immature cell bodies, i.e., the cortical plate. The band dissipated as this tier differentiated postnatally into the supragranular layers. Electron microscopy showed that the supragranular layers, which contain the highest density of noradrenergic fibers, also contain the highest areal density of labeled postsynaptic junctions beyond 2 weeks of age. Throughout the ages, the majority of immunoreactivity occurred at sites which, in single ultrathin sections, appeared to be nonjunctional sites of axons, dendrites, and in glial processes. Our observations indicate that (1) both pyramidal and nonpyramidal neurons are receptive to norepinephrine via alpha 2AAR, (2) alpha 2AAR synthesis is robust prior to synaptogenesis, and (3) alpha 2AAR operates both pre- and postsynaptically.

Animals↗
PubMed1994

Sequestration of muscarinic acetylcholine receptor m2 subtypes. Facilitation by G protein-coupled receptor kinase (GRK2) and attenuation by a dominant-negative mutant of GRK2.

Sequestration of m2 receptors (muscarinic acetylcholine receptor m2 subtypes), which was assessed as loss of N-[3H]methylscopolamine ([3H]NMS) binding activity from the cell surface, was examined in COS 7 and BHK-21 cells that had been transfected with expression vectors encoding the m2 receptor and, independently, vectors encoding a G protein-coupled receptor kinase (GRK2) (beta-adrenergic receptor kinase 1) or a GRK2 dominant-negative mutant (DN-GRK2). The sequestration of m2 receptors became apparent when the cells were treated with 10(-5) M or higher concentrations of carbamylcholine. In this case, approximately 40% or 20-25% of the [3H]NMS binding sites on COS 7 or BHK-21 cells, respectively, were sequestered with a half-life of 15-25 min. In cells in which GRK2 was also expressed, the sequestration became apparent in the presence of 10(-7) M carbamylcholine. Approximately 40% of the [3H]NMS binding sites on both COS 7 and BHK-21 cells were sequestered in the presence of 10(-6) M or higher concentrations of carbamylcholine. When DN-GRK2 was expressed in COS 7 cells, the proportion of [3H]NMS binding sites sequestered in the presence of 10(-5) M or higher concentrations of carbamylcholine was reduced to 20-30%. These results indicate that the phosphorylation of m2 receptors by GRK2 facilitates their sequestration. These results are in contrast with the absence of a correlation between sequestration and the phosphorylation of beta-adrenergic receptors by the GRK2 and suggests that the consequences of phosphorylation by GRK2 are different for different receptors.

Animals↗
PubMed1994

Determination of the G beta gamma-binding domain of phosducin. A regulatable modulator of G beta gamma signaling.

Although a role for the beta gamma-subunits of heterotrimeric G proteins (G beta gamma) in signal transduction by several cellular systems has been established, the structural features of cellular proteins interacting with G beta gamma have yet to be fully elucidated. The G beta gamma-binding region of beta-adrenergic receptor kinase (beta ARK), a cytosolic enzyme recruited to the membrane receptor substrate by G beta gamma, has been localized to the carboxyl terminus of the enzyme. Here, we demonstrate that the amino terminus of phosducin, a 33-kDa G beta gamma-binding retinal phosphoprotein, contains sequences homologous with the G beta gamma-binding domain of beta ARK. Accordingly, a glutathione S-transferase-fusion protein containing only the amino-terminal 105 amino acids of phosducin displayed G beta gamma binding ability. This domain of phosducin contains a protein kinase A (PKA) phosphorylation site, and upon phosphorylation, the binding of full-length phosducin to G beta gamma is reduced. In addition, transient expression of phosducin in COS-7 cells significantly inhibits G beta gamma-mediated phosphoinositide hydrolysis. This inhibitory effect is completely reversed by pretreatment of cells with dibutyryl cAMP, an activator of PKA. Thus, the binding of G beta gamma to phosducin can be regulated by PKA-phosphorylation in an intact cell model system.

Amino Acid Sequence↗
PubMed1994

Perikaryal and synaptic localization of alpha 2A-adrenergic receptor-like immunoreactivity.

Through molecular cloning, the existence of three distinct subtypes of alpha 2-adrenergic receptors (alpha 2AR)--A, B and C--has been established and are referred to as alpha 2A AR, alpha 2B AR and alpha 2CAR. Due to limitations in pharmacological tools, it has been difficult to ascribe the role of each subtype to the central functions of alpha 2AR. In situ hybridization studies have provided valuable information regarding their distribution within brain. However, little is known about their subcellular distribution, and in particular, their pre- versus postsynaptic localization or their relation to noradrenergic neurons in the CNS. We used an antiserum that selectively recognizes the A-subtype of alpha 2AR to determine: (1) the regional distribution of the receptor within brains of rat and monkey; (2) the subcellular distribution of the receptor in locus coeruleus (LC) of rats and prefrontal cortex of monkeys; and (3) the ultrastructural relation of the receptor to noradrenergic processes in LC. Light microscopic immunocytochemistry revealed prominent immunoreactivity in LC, the brainstem regions modulating the baroreflex, the granule cell layer of the cerebellar cortex, the paraventricular and supraoptic nuclei of the hypothalamus (PVN, SON), the basal ganglia, all thalamic nuclei, the hippocampal formation and throughout cerebral cortical areas. Comparison of results obtained from rat and monkey brains revealed no apparent interspecies-differences in the regional distribution of immunoreactivity. Immunoreactivity occurred as small puncta, less than 1 micron in diameter, that cluster over neuronal perikarya. Besides these puncta, cell bodies, proximal dendrites and fine varicose processes--most likely to be axonal--of the PVN and SON and the hippocampal granule cells also exhibited homogeneously intense distribution of immunoreactivity. Subcellularly, alpha 2AAR-ir in LC and prefrontal cortex were associated with synaptic and non-synaptic plasma membrane of dendrites and perikarya as well as perikaryal membranous organelles. In addition, cortical tissue, but not LC, exhibited prominent immunoreactivity within spine heads. Rat brainstem tissue immunolabeled dually for alpha 2AAR and dopamine beta-hydroxylase (D beta H, the noradrenaline-synthesizing enzyme) revealed that alpha 2AAR-li occurs in catecholaminergic terminals but is also prevalent within non-catecholaminergic terminals. Terminals exhibiting alpha 2AAR-li formed symmetric and asymmetric types of synapses onto dendrites with and without D beta H-immunoreactivity. These results indicate that: (1) the A-subtype of alpha 2AR is distributed widely within brain; (2) alpha 2AAR-li reflects the presence of newly synthesized alph 2AAR in perikarya as well as those receptors along the plasma membrane of perikarya, dendritic trunks and spines; and (3) alpha 2AAR in LC may operate as heteroreceptors on non-catecholaminergic terminals as well as autoreceptors on noradrenergic terminals.

Animals↗
PubMed1994

Differential desensitization and phosphorylation of three cloned and transfected alpha 2-adrenergic receptor subtypes.

Genes encoding 3 distinct subtypes of human alpha 2-adrenergic receptor are known and are found, respectively, on chromosome 10, 4, and 2 (alpha 2-C10, alpha 2-C4, and alpha 2-C2 adrenergic receptors). All 3 receptors inhibit adenylyl cyclase via Gi proteins. To study and compare their regulatory properties we assessed the ability of each to undergo agonist-promoted desensitization and phosphorylation. When Chinese hamster ovary cells stably expressing each of the three receptor genes were incubated with epinephrine for 20 min, a marked decrease in sensitivity to subsequent agonist-mediated inhibition of adenylyl cyclase was observed for the alpha 2-C10 and alpha 2-C2 receptors but not for the alpha 2-C4 receptors. When similar incubations were performed with 32Pi-labeled cells and the receptors were immunoprecipitated with specific antibodies, alpha 2-C10 and alpha 2-C2 receptors were found to undergo an approximately 3-fold increase in receptor phosphorylation after epinephrine exposure. When transfected into COS cells epinephrine also stimulated phosphorylation of alpha 2-C10 and alpha 2-C2 receptors while having only a slight effect on alpha 2-C4 receptors. Cotransfection of the cells with the cDNA encoding the beta-adrenergic receptor kinase further increased receptor phosphorylation for alpha 2-C10 and alpha 2-C2 receptors while having little or no effect on alpha 2-C4 receptors. Moreover purified and reconstituted recombinant alpha 2-C10 receptors could be phosphorylated in an agonist-dependent fashion whereas alpha 2-C4 receptors could not. These observations suggest receptor subtype-specific differences in susceptibility to regulatory phosphorylation and desensitization.

Adenylyl Cyclase Inhibitors↗
PubMed1994

Similar ligand binding in recombinant human alpha 2 C2-adrenoceptors produced in mammalian, insect and yeast cells.

Ligand binding properties were investigated in recombinant human alpha 2C2-adrenoceptors expressed in three different host systems: Shionogi S115 mouse mammary tumour cells, Spodoptera frugiperda Sf9 insect cells and Saccharomyces cerevisiae yeast cells. The expected 43 kDa alpha 2C2 protein was visualized with immunoblotting using a polyclonal alpha 2C2-receptor antibody. [3H]Rauwolscine binding in cell homogenates or membranes (Bmax 3-11 pmol/mg protein; Kd approximately 5.5 nM) was inhibited by prazosin, oxymetazoline, RX821002, chlorpromazine and (-)-noradrenaline with and without the GTP-analogue Gpp(NH)p with similar Ki values in the different host systems. This indicates that alpha 2C2-adrenoceptors retain their binding characteristics irrespective of the host environment.

Adrenergic alpha-Antagonists↗
PubMed1994

[Regulation of G proteins by receptors].

Receptors located on the cell surface are responsible for recognition of extracellular first messengers. The largest number of receptors belongs to a G protein-coupled receptor superfamily. They showed common structural features characterized by seven transmembrane regions and three connecting extracellular and intracellular loops. Stimulation of a receptor by an agonist activates the coupled G protein. From in vitro mutagenesis analyses of beta 2-adrenergic, muscarinic acetylcholine and alpha 1-adrenergic receptors, it was shown that the intracellular third loop and carboxyl terminus of the receptor molecule are involved in coupling with a G protein. One intriguing observation was that mutation at a single site of the intracellular third loop could induce the active state of receptors without agonist stimulation. Receptor heterogeneity generated from distinct genes or alternative splicing can be seen at the intracellular third loop and carboxyl terminus that is assumed to play an important role of coupling with G proteins. There are several examples that receptor isoforms arising from alternative splicing have their own counterparts of G proteins.

Amino Acid Sequence↗
PubMed1993

Constitutively active mutants of the alpha 2-adrenergic receptor.

We have mutated a single residue, Thr373 [corrected], in the C-terminal portion of the third intracellular loop of the alpha 2C10-adrenergic receptor into five different amino acids. In analogy with the effect of similar mutations in the alpha 1B- and beta 2-adrenergic receptors, these substitutions resulted in two major biochemical modifications: 1) increased constitutive activity of the alpha 2-adrenergic receptor leading to agonist-independent inhibition of adenylyl cyclase and 2) increased affinity of the receptor for binding agonist but not antagonists. The increased constitutive activity of the mutated alpha 2-adrenergic receptors could be inhibited by pertussis toxin, clearly indicating that it results from spontaneous ligand-independent receptor coupling to Gi. In contrast, the increased affinity of the mutant receptors for binding agonists was unaffected by pertussis toxin treatment, indicating that this is an inherent property of the receptors not dependent on interaction with Gi. Coexpression of the receptor mutants with the receptor-specific kinase, beta ARK1, indicated that the constitutively active alpha 2-adrenergic receptors are substrates for beta-adrenergic receptor kinase (beta ARK)-mediated phosphorylation even in the absence of agonist. These findings strengthen the idea that constitutively active adrenergic receptors mimic the "active" state of a G protein-coupled receptor adopting conformations similar to those induced by agonist when it binds to wild type receptors. In addition, these results extend the notion that in the adrenergic receptor family the C-terminal portion of the third intracellular loop plays a general role in the processes involved in receptor activation.

Adenylate Cyclase Toxin↗
PubMed1993

Characterization of alpha 2-adrenergic receptor subtype-specific antibodies.

Subtypes of alpha 2-adrenergic receptors have been defined pharmacologically in a variety of mammalian tissues. The alpha 2A, alpha 2B, alpha 2C, and most recently alpha 2D subtypes have been characterized by their affinities for selective receptor antagonists and agonists. The genes that may encode the alpha 2A, alpha 2B, and alpha 2C subtypes have been identified in human and rat. In human these genes are termed alpha 2-C10, alpha 2-C2, and alpha 2-C4, respectively, based on their chromosomal localization, whereas three genes, designated RG20 alpha 2, RNG alpha 2, and RG10 alpha 2, are thought to be the corresponding rat homologues. These assignments were based on the pharmacology of the cloned receptor genes expressed in transfected cells and on the detection of homologous mRNAs by Northern blot analyses in cell lines or tissues with pharmacologically defined alpha 2-adrenergic receptors. However, the subtype assignment of cloned genes has not been fully resolved by these means. To help clarify the subtype assignment, we have raised antibodies against sequences from the divergent third intracellular loop of the human and rat alpha 2-adrenergic receptors. These antibodies were found to be subtype specific in immunoprecipitating either the cloned receptors expressed by DNA transfection or the pharmacologically defined receptors prepared from various tissues. Our immunological data corroborate the assignments of alpha 2-C2/RNG alpha 2 as encoding the alpha 2B subtype in NG108-15 cells and rat neonatal lung and of alpha 2-C4/RG10 alpha 2 as encoding the alpha 2C subtype in opossum kidney cells. Furthermore, antibodies against alpha 2-C10 and RG20 alpha 2 but not alpha 2-C2/RNG alpha 2 or alpha 2-C4/RG10 alpha 2 were both found to recognize alpha 2-adrenergic receptors expressed in rat submaxillary glands and in bovine pineal gland, two tissues with alpha 2D pharmacology. Because three genes were identified in the rat and human genome, these data suggest that the pharmacologically defined "alpha 2D receptor" is genetically of the alpha 2A subtype.

Adrenergic alpha-Agonists↗
PubMed1992

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.

Adenosine Diphosphate Ribose↗
PubMed1992

Tyrosine phosphorylation of G protein alpha subunits by pp60c-src.

A number of lines of evidence suggest that cross-talk exists between the cellular signal transduction pathways involving tyrosine phosphorylation catalyzed by members of the pp60c-src kinase family and those mediated by guanine nucleotide regulatory proteins (G proteins). In this study, we explore the possibility that direct interactions between pp60c-src and G proteins may occur with functional consequences. Preparations of pp60c-src isolated by immunoprecipitation phosphorylate on tyrosine residues the purified G-protein alpha subunits (G alpha) of several heterotrimeric G proteins. Phosphorylation is highly dependent on G-protein conformation, and G alpha(GDP) uncomplexed by beta gamma subunits appears to be the preferred substrate. In functional studies, phosphorylation of stimulatory G alpha (G alpha s) modestly increases the rate of binding of guanosine 5'-[gamma-[35S]thio]triphosphate to Gs as well as the receptor-stimulated steady-state rate of GTP hydrolysis by Gs. Heterotrimeric G proteins may represent a previously unappreciated class of potential substrates for pp60c-src.

Animals↗