Stimulatory Guanine Nucleotide-binding Regulatory Protein Research Articles

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.

Sensitization of C-fibres by prostaglandin E2 in the rat is inhibited by guanosine 5'-O-(2-thiodiphosphate), 2',5'-dideoxyadenosine and Walsh inhibitor peptide.

Behavioral studies have shown that mechanical hyperalgesia induced by intradermal injection of prostaglandin E2 is blocked by inhibitors of the cAMP second messenger system. Similarly, injection of prostaglandin E2 also induces a decrease in mechanical threshold and an increase in the number of action potentials elicited by test stimuli in most C-fibre nociceptors. This change is called sensitization. To further evaluate the degree of correlation between primary afferent sensitization and mechanical hyperalgesia, we conducted a study to evaluate the effect of agents known to block the cAMP second messenger system and behavioral manifestations of mechanical hyperalgesia following injection of prostaglandin E2. The agents tested were guanosine 5'-O-(2-thiodiphosphate), an inhibitor of stimulatory guanine nucleotide-binding regulatory proteins; 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase; and Walsh Inhibitor Peptide, an inhibitor of cAMP-dependent protein kinase. Single fibre electrophysiologic studies of 138 C-fibres, innervating the dorsum of the hind paw, was done in male Sprague-Dawley rats. The number of spikes evoked by a 10 s application of a threshold von Frey hair were determined before and after intradermal injection of test agents administered alone and in combination with prostaglandin E2. Injection of prostaglandin E2 with the test agent vehicle (saline or distilled water) resulted in a significant decrease in von Frey hair threshold and an increase in the number of spikes generated in response to threshold von Frey hairs. In contrast, co-injection of prostaglandin E2 with guanosine-5'-O-(2-thiodiphosphate), 2',5'-dideoxyadenosine or Walsh inhibitor peptide did not result in a significant decrease in von Frey hair mechanical threshold or increase in the number of spikes generated to the threshold stimuli, compared with vehicle/prostaglandin E2. It is suggested that guanosine 5'-O-(2-thiodiphosphate), 2',5'-dideoxyadenosine and Walsh inhibitor protein inhibited prostaglandin E2 sensitization of primary afferent C-fibres by inhibiting a stimulatory guanine nucleotide-binding regulatory protein, adenylyl cyclase, and protein kinase A, respectively. These results support the hypothesis that primary afferent sensitization by prostaglandin E2 underlies prostaglandin E2-induced hyperalgesia.

Multiple GTP-binding proteins in sea urchin sperm: Evidence for Gs and small G-proteins.

Sea urchin sperm plasma membranes isolated from heads and flagella were used to examine the presence of Gs (stimulatory guanine nucleotide-binding regulatory protein) and small G-proteins. Flagellar plasma membranes incubated with [32 P]NAD and cholera toxin (CTX) displayed radiolabeling in a protein of 48 kDa, which was reactive by immunoblotting with a specific antibody against mammalian Gs. CTX-catalyzed [32 P]ADP-ribosylation in conjunction with immunoprecipitation with anti-Gs, followed by electrophoresis and autoradiography, revealed one band of 48 kDa. Head plasma membranes, in contrast, did not show substrates for ADP-ribosylation by CTX. In flagellar and head plasma membranes pertussis toxin (PTX) ADP-ribosylated the same protein described previously in membranes from whole sperm; the extent of ADP-ribosylation by PTX was higher in flagellar than in head membranes. Small G-proteins were investigated by [32 P]GTP-blotting. Both head and flagellar plasma membranes showed three radiolabeled bands of 28, 25 and 24 kDa. Unlabeled GTP and GDP, but not other nucleotides, interfered with the [α-32 P]GTP-binding in a concentration-dependent manner. A monoclonal antibody against human Ras p21 recognized a single protein of 21 kDa only in flagellar membranes. Thus, sea urchin sperm contain a membrane protein that shares characteristics with mammalian Gs and four small G-proteins, including Ras. Gs, Gi and Ras are enriched in flagellar membranes while the other small G-proteins do not display a preferential distribution along the sea urchin sperm plasma membrane. The role of these G-proteins in sea urchin sperm is presently under investigation.

Is there more than one prostaglandin E receptor subtype mediating hyperalgesia in the rat hindpaw?

Five synthetic prostaglandin E analogs (11-deoxyPGE1, 17-phenyl-ol-trinor prostaglandin E2, enisoprost, MB28767 and misoprostol) have been evaluated for their ability to produce mechanical hyperalgesia in rats. The Randall-Selitto paw withdrawal model of mechanical hyperalgesia was used. Following intradermal injections (2.5 microliters) into the dorsal surface of the hindpaw, each prostaglandin E analog produced a dose-dependent (1-1000 ng) decrease in nociceptive threshold (i.e. hyperalgesia). Hyperalgesia produced by 17-phenyl-ol-trinor prostaglandin E2 and MB28767, was inhibited by the prostaglandin E1 antagonist SC19220 (7.5 ng), while the hyperalgesia produced by 11-deoxyprostaglandin E1, enisoprost and misoprostol was not inhibited by this antagonist. Hyperalgesia produced by all five analogs was significantly attenuated or completely blocked by inhibiting stimulatory guanine nucleotide-binding regulatory protein with guanosine 5'-O-(2-thiodiphosphate), adenylyl cyclase with 2'5'-dideoxyadenosine and protein kinase A with WIPTIDE. These results suggest the presence of more than one prostaglandin E-receptor subtype, which mediate hyperalgesia, predominantly via the cAMP second messenger system, in the hindpaw of the rat.

Comparison of prostaglandin E 1- and prostaglandin E 2-induced hyperalgesia in the rat

We have studied prostaglandin E 1-induced mechanical hyperalgesia in the rat hindpaw, by assessing paw-withdrawal thresholds, before and after injecting prostaglandin E 1 alone or with other agents, in normal and streptozotocin-induced diabetic rats. In normal and diabetic rats, prostaglandin E 1 (1–1000 ng) produced a dose-dependent decrease in mechanical nociceptive threshold. In diabetic rats, prostaglandin E 1 was more potent than in normal rats, in producing hyperalgesia, whereas prostaglandin E 2 hyperalgesia was not changed in normal and diabetic rats. Prostaglandin Ei-induced hyperalgesia was not inhibited by E-type 1 prostaglandin receptor antagonists, SC 19220 or SC51089, either in normal or diabetic rats. In fact, in the presence of SC19220, prostaglandin E 1 produced enhanced hyperalgesia, in normal rats. Prostaglandin E 1 hyperalgesia was not significantly modified by sympathectomy or indomethacin. Unlike prostaglandin E 2, prostaglandin E 1 hyperalgesia was not blocked by the inhibitor of the stimulatory guanine nucleotide-binding regulatory protein, guanosine 5'-O-(2-thiodiphosphate). It is suggested that prostaglandin E 1 decreases primary afferent nociceptive threshold directly, by activating a prostaglandin receptor other than the E-type 1 prostaglandin receptor, and that this receptor is not coupled to a stimulatory guanine nucleotide-binding regulatory protein.

Interaction between a phorbol ester and dopamine DA1 receptors on vascular smooth muscle

The interaction between dopamine DA1 receptors and a phorbol ester was studied to elucidate the role of protein kinase C in the response of this receptor. The in vitro binding of [3H]Sch 23390 to DA1 receptor sites on vascular smooth muscle cells was saturable. The extent of [3H]Sch 23390 binding to phorbol ester-treated cells was increased without any change in the dissociation constant. The production of adenosine 3',5'-cyclic monophosphate (cAMP) in response to DA1 receptor stimulation was enhanced by preincubation of vascular smooth muscle cells with the phorbol ester for 4 h. However, no enhancement was observed when the medium used for preincubation was supplemented with a protein kinase C inhibitor. Direct stimulation of stimulatory guanine nucleotide-binding regulatory protein with 5-guanylylimidodiphosphate and direct stimulation of adenylate cyclase with forskolin produced no significant differences in cyclase levels between phorbol ester-treated and untreated cells. These results suggest that activation of protein kinase C triggers an increase in the membrane expression of DA1 receptors, thereby enhancing receptor-coupled cAMP generation.

Regulation of cyclic adenosine monophosphate synthesis in human ejaculated spermatozoa. I. Experimental conditions to quantitate membrane-bound adenylyl cyclase activity.

Although cyclic adenosine monophosphate (cAMP) is an important regulator of motility and metabolism in human spermatozoa, little is known on the cellular system responsible for its synthesis. Here, we investigated the experimental conditions directly to quantitate adenylyl cyclase (AC) activity synthesizing cAMP in human ejaculated spermatozoa and analysed the general properties of the enzyme. A 10,000 g membrane fraction was prepared from washed sperm cells homogenized by sonication. AC activity was monitored by the direct conversion of [alpha-32P]adenosine triphosphate (ATP) into [32P]cAMP. Using a nucleoside triphosphate regenerating system to ensure availability of ATP substrate, the human sperm AC showed a steady production of cAMP for at least 1 h. The assay was optimized for pH, buffer concentration, membrane protein and substrate concentration. Activity was dependent upon the presence of Mn2+ as a divalent cation and showed a pH optimum between 7.0 and 8.5. Optimal activity required 5 mM ATP, 1 mM ethylenediamine tetraacetic acid (EDTA) and 20-40 mM total MnCl2. Dependence on Mn2+ was not mandatory; Mg2+ at 5-40 mM also supported significant activity, but the activity was 4-6 times lower than that with Mn2+. Regardless of the presence of Mn2+ or Mg2+ as divalent cation in the assay, human sperm AC was insensitive to the regulatory ligands NaF, guanine nucleotide or forskolin. Insensitivity to these ligands supports the proposal that this enzyme system does not contain a stimulatory guanine nucleotide-binding regulatory protein and that its catalytic component is unique and different from that of somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of the recombinant spliced variants of the α-sub-unit of the stimulatory guanine-nucleotide binding regulatory protein (G s) by the catalytic sub-unit of protein kinase a

Both G sα-1 and G sα-4 were phosphorylated by the purified catalytic sub-unit of protein kinase A. Phosphate incorporation into 220 pmol and 190 pmol of G sα-4 and G sα-1 after a 1 hour incubation with kinase was 14 pmol and 10 pmol, respectively. These low levels of phosphorylation are due to the thermal lability of purified recombinant G sα. However, the phosphorylation was inhibited by guanine nucleotides (GDP-β-S, GppNHp and GTP) and is, therefore, a specific event. We suggest that, as for G sα phosphorylation by protein kinase C (Pyne et al., 1992), the guanine nucleotide-free form of G sα is the most likely substrate. Guanine-nucleotides reduce the lifetime and, therefore availability for phosphorylation, of guanine-nucleotide free G sα. G sα phosphorylation by protein kinase A in vitro provides preliminary evidence that a similar phosphorylation of G sα may be an important regulatory event in cells.

The role of Mg2+ on the formation of the ternary complex between agonist, beta-adrenoceptor, and GS-protein and an interpretation of high and low affinity binding of beta-adrenoceptor agonists.

In the absence of GTP or its analogues, the beta-adrenoceptor agonist (H), the receptor (R) and the stimulatory guanine nucleotide-binding regulatory protein (GS-protein) form a slowly dissociable complex (HRGSGDP). It is presumed that the agonist in this ternary complex is bound to the receptor with high affinity while its binding to the receptor in the HR-complex is of low affinity. We have studied the effect of Mg2+ on the formation of these two complexes by the displacement of (-)-[3H]CGP-12177 with (-)-isoprenaline in cell membranes prepared from guinea-pig lung parenchyma. In the absence of Mg2+, only monophasic displacement curves were obtained with a dissociation constant not differing from that obtained in the presence of Gpp(NH)p and Mg2+. In the presence of Mg2+ and absence of Gpp(NH)p, a shallow binding curve was obtained that fitted a two site model best. One of the dissociation constants agreed well with that obtained in the presence of Gpp(NH)p and Mg2+ but the other one was 27 times smaller. It was concluded that Mg2+ is necessary for the coupling between the HR-complex and the GS-protein. The two dissociation constants obtained in the absence of Gpp(NH)p represent the dissociation constants of the HR- and HRGSGDP-complexes and may correspond to those named KL and KH in the literature. According to our study, there may be no difference in affinity between the agonist and receptor in the HR- and HRGSGDP-complexes. The two dissociation constants obtained in the absence of GTP or its analogues refer to two consecutive reactions.

Regulation of prostaglandin E2 receptor binding activity in porcine temporal cortex by protein phosphorylation.

Regulation of prostaglandin (PG) E2 receptors was investigated in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-solubilized fraction from the synaptic membrane of porcine temporal cortex. The fraction was preincubated with exogenous protein kinases, and then the binding of PGE2 was measured. PGE2 binding was increased approximately twofold by pretreatment with the catalytic subunit of cyclic AMP-dependent protein kinase (A kinase) or calmodulin-dependent protein kinase II but not by that with protein kinase C. The increase was dependent on the ATP concentration, with ED50 values being close to the Km values of these protein kinases. Protein kinase inhibitors specific for A kinase and for calmodulin-dependent protein kinase II abolished the effect in a dose-dependent manner, with IC50 values being similar to those reported. Further study using the catalytic subunit of A kinase revealed that the maximal binding capacity apparently increased without affecting the affinity and the rate constants for association and dissociation. On the other hand, acid phosphatase treatment reduced the binding activity to the level of nonspecific binding. In addition, treatment by A kinase did not affect the binding of guanosine 5'-(3-thiotriphosphate) by the GTP-binding proteins and the activation of adenylate cyclase mediated by stimulatory guanine nucleotide-binding regulatory protein, and therefore the phosphorylation is believed to occur on the receptor protein. The results suggest that the PGE2 receptor can take active phosphorylated and inactive dephosphorylated forms, of which only the phosphorylated one can bind PGE2.

Glucagon induces disaggregation of polymer-like structures of the alpha subunit of the stimulatory G protein in liver membranes.

The hydrodynamic behavior of G alpha s, the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein (G protein), in octyl glucoside extracts of rat liver membranes was investigated. As was previously shown for G proteins similarly extracted from brain synaptoneurosomes, G alpha s behaved as polydisperse structures with S values higher than that of heterotrimeric G proteins. At concentrations of guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) greater than 100 microM, incubation with membranes led to smaller structures having S values in the range of 4-5 S. Incubation of liver membranes with glucagon also caused a marked increase in structures having these S values; glucagon action required the presence of low concentrations of GTP[gamma S] (maximal, 10 microM), was rapid (within 10 sec), and was not observed with vasopressin, angiotensin II, or glucagon-(19-29). When G alpha s in its membrane-bound form was [32P]ADP-ribosylated by cholera toxin and the treated membranes were extracted with octyl glucoside, greater than 35% of the labeled G alpha s was found in material that sedimented through sucrose gradients and contained relatively low levels of immunoreactive G alpha s. Glucagon selectively converted the apparently large molecular weight structures to the 4-5 S structures in the presence of GTP[gamma S], even at 1 mM (the maximal effect of the nucleotide alone), when incubated with the toxin-treated membranes. These findings suggest that the glucagon receptor selectively interacts with polymer-like structures of G alpha s and that activation by GTP[gamma S] results in disaggregation. The role of the beta and gamma subunits of G proteins in the hormone-induced process is not clear since the polymer-like structures extracted with octyl glucoside are devoid of beta and gamma subunits.

Altered β Adrenergic Receptor Function in Subjects with Symptomatic Mitral Valve Prolapse

Individuals with mitral valve prolapse (MVP) frequently show symptoms of a hyperadrenergic state. beta adrenergic receptor characteristics were compared in the lymphocytes of subjects with symptomatic MVP and control subjects during rest and exercise. At rest, the proportion of receptors binding agonist with high affinity, as determined from isoproterenol competition for (-)[125I]-iodopindolol binding sites, was greater in MVP subjects than in controls. With exercise, the proportion of high-affinity receptors in MVP subjects decreased to control levels. Isoproterenol stimulation of lymphocyte 3',5'-cyclic adenosine monophosphate (cyclic AMP) also was greater in MVP subjects than in controls at rest, but not during exercise. Plasma catecholamine concentrations in MVP subjects were normal during both rest and exercise. Unlike exercise, isoproterenol infusion elicited clinical manifestations of increased adrenergic responsiveness in MVP subjects. The beta receptor in exercised MVP subjects exhibited unusually high affinity agonist binding (i.e. a lower dissociation constant KH than in either the same subjects at rest or exercised controls) and also abnormal coupling to the stimulatory guanine nucleotide-binding regulatory protein (GS) of adenylate cyclase, as reflected by the inability of guanine nucleotide to convert the receptor to a low-affinity state. These findings suggest that functional alteration of the beta adrenergic receptor, in the absence of abnormal plasma catecholamine levels, might contribute to the hyperadrenergic state of MVP subjects at rest. However, desensitization of high affinity beta receptors or altered receptor-GS coupling might preserve normal adrenergic responsiveness during exercise.

Effects of long-term treatment with lithium and antidepressants on [ 3H]forskolin binding to rat cerebral cortical membranes

Odagaki, Yuji, Tsukasa Koyama and Itaru Yamashita: Effects of long-term treatment with lithium and antidepressants on [ 3H] forskolin binding to rat cerebral cortical membranes. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1991, 15 (5): 709–716. 1. 1. The characteristics of [ 3H]forskolin binding in rat cerebral cortical membranes and the effects of long-term treatment with lithium, desipramine and tranylcypromine on the binding were investigated. 2. 2. Specific binding of [ 3H] forskolin (10 nM) was augmented by the addition of magnesium, and in the presence of 5 mM MgCl 2, sodium fluoride (NaF) increased further the specific binding in a concentration-dependent manner. 3. 3. Guanyl-5'-ylimidodiphosphate (Gpp(NH)p) increased the specific binding only in the presence of 100 mM MgCl 2. 4. 4. Long-term treatment with lithium, desipramine or tranylcypromine altered neither the specific binding of [ 3H] forskolin nor the augmentation by MgCl 2, NaF, or Gpp(NH)p. 5. 5. The results indicate that these preparations alter neither the catalytic subunit of adenylate cyclase nor the activation of stimulatory guanine nucleotide-binding regulatory proteins (Gs).

Opposing Effects of Ethanol on Pig Ovarian Adenylyl Cyclase Desensitized by Human Choriogonadotropin or Isoproterenol*

Pig ovarian follicular membranes contain a gonadotropin-responsive adenylyl cyclase, which becomes partially desensitized (approximately 40%) upon a 40-min incubation with a saturating concentration of human (h) CG. This in vitro desensitization is time and hormone dependent and also requires the presence of micromolar concentrations of GTP. In this report we show that 10% ethanol present during the desensitization phase of the incubation increases the extent of hCG-induced desensitization of adenylyl cyclase by 2-fold. Ethanol shortened the time necessary to reach maximal hCG-induced desensitization from 20 to 10 min, but had no effect on the dose dependency for GTP. In addition, ethanol had no effect on the affinity of the LH/hCG receptor for 125I-hCG but did cause an increase in the ED50 of hCG for inducing desensitization from 0.25 to 0.75 nM. Interestingly, ethanol decreased the apparent number of LH/hCG-receptor sites by 55%, yet the control hCG-sensitive adenylyl cyclase activity was not reduced. The "hyperdesensitized" state achieved in the presence of ethanol could not be reversed by washing the membranes and incubating them in ethanol-free medium. NaF-sensitive adenylyl cyclase was also not impaired in hCG-desensitized membranes from control or ethanol-treated samples. Thus, hCG-induced desensitization was not due to a defect in the functioning of the stimulatory guanine nucleotide-binding regulatory protein (G8) or catalytic subunits, but rather was caused by an impairment of the coupling of the lutropin (LH)/hCG receptor with G8, which was exacerbated further by ethanol. In spite of the effect of ethanol on hCG-induced desensitization, this agent had an inhibitory effect on isoproterenol-induced desensitization of isoproterenol-responsive luteal adenylyl cyclase. These results indicate that membrane fluidity is important in modulating the structure and functional interaction of the LH/hCG receptor with G8 because ethanol is a well known lipid-fluidizing agent. The resistance to ethanol potentiation of desensitization of the isoproterenol-sensitive adenylyl cyclase suggests that there are differences between the LH/hCG- and beta-adrenergic receptors in factors controlling their structures and or interactions with G proteins, and that there is a fundamental difference in their mechanisms of desensitization.

Agonist-induced ADP-ribosylation of a cytosolic protein in human platelets.

alpha-Thrombin and phorbol 12,13-dibutyrate stimulated the mono(ADP-ribosyl)ation of a 42-kDa cytosolic protein of human platelets. This effect was mediated by protein kinase C activation and was inhibited by protein kinase C inhibitor staurosporine. It also was prevented by prostacyclin, which is known to inhibit the phospholipase C-induced formation of 1,2-diacylglycerol, which is one of the endogenous activators of protein kinase C. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the 42-kDa protein that is ADP-ribosylated by alpha-thrombin was clearly distinct from the alpha subunits of membrane-bound inhibitory and stimulatory guanine nucleotide-binding regulatory proteins, respectively Gi alpha and Gs alpha; the 47-kDa protein that is phophorylated by protein kinase C in platelets; and the 39-kDa protein that has been shown to be endogenously ADP-ribosylated by agents that release nitric oxide. This information shows that agonist-induced activation of protein kinase leads to the ADP-ribosylation of a specific protein. This covalent modification might have a functional role in platelet activation.

Multiple Effects of Ethanol on Cardiac Adenylate Cyclase

Summary: We characterized the effects of ethanol on the activators of adenylate cyclase complex that act through the receptor site, the stimulatory guanine nucleotidebinding regulatory protein (Gs), or the catalytic unit. Ethanol had no effect on adenylate cyclase activity stimulated by Mn2+, a selective activator of the catalytic unit, whereas high concentrations of ethanol inhibited both basal and isoproterenol-stimulated adenylate cyclase. In contrast, in the presence of nonhydrolyzable GTP analogs, ethanol potentiated substantial increases in adenylate cyclase activity. In the presence of these GTP analogs, ethanol increased the Vmax without altering the affinity of adenylate cyclase for ATP. Ethanol also increased adenylate cyclase activity in membranes in which Gs had been preactivated with isoproterenol plus a nonhydrolyzable GTP analog, suggesting that ethanol enhanced the interaction between activated Gs and the catalytic unit. Paradoxically, the ability of cholera toxin and NAD+ to augment adenylate cyclase activity through an effect on Gs was attenuated by increasing concentrations of ethanol. These results suggest that acute exposure to ethanol has multiple effects on cardiac membrane adenylate cyclase.

Multiple Effects of Ethanol on Cardiac Adenylate Cyclase

We characterized the effects of ethanol on the activators of adenylate cyclase complex that act through the receptor site, the stimulatory guanine nucleotide-binding regulatory protein (Gs), or the catalytic unit. Ethanol had no effect on adenylate cyclase activity stimulated by Mn2+, a selective activator of the catalytic unit, whereas high concentrations of ethanol inhibited both basal and isoproterenol-stimulated adenylate cyclase. In contrast, in the presence of nonhydrolyzable GTP analogs, ethanol potentiated substantial increases in adenylate cyclase activity. In the presence of these GTP analogs, ethanol increased the Vmax without altering the affinity of adenylate cyclase for ATP. Ethanol also increased adenylate cyclase activity in membranes in which Gs had been preactivated with isoproterenol plus a nonhydrolyzable GTP analog, suggesting that ethanol enhanced the interaction between activated Gs and the catalytic unit. Paradoxically, the ability of cholera toxin and NAD+ to augment adenylate cyclase activity through an effect on Gs was attenuated by increasing concentrations of ethanol. These results suggest that acute exposure to ethanol has multiple effects on cardiac membrane adenylate cyclase.

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein.

ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5'-[alpha-32P]triphosphate in the cytosol and membranes. These findings indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an alpha subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.

Site-directed mutagenesis of human beta-adrenergic receptors: substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase.

By using oligonucleotide-directed mutagenesis, we have produced a point mutation (guanine to adenine) at nucleotide 388 of the gene for human beta-adrenergic receptor (beta AR) that results in a substitution of asparagine for the highly conserved aspartic acid at position 130 in the putative third transmembrane domain of the human beta AR ([Asn130]beta AR). We have examined the functional significance of this mutation in B-82 cells continuously expressing the mutant [Asn130]beta AR. The mutant [Asn130]beta AR displayed normal antagonist binding but unusually high-affinity agonist binding (5- to 10-fold higher than wild-type beta AR), consistent with a single class of high-affinity binding sites. The mutant beta AR displayed guanine nucleotide-sensitive changes in agonist affinity (3- to 5-fold shift) implying an interaction between the beta AR and the stimulatory guanine nucleotide-binding regulatory protein; however, the ability of guanine nucleotides to alter agonist affinity was attenuated. Addition of saturating concentrations of isoproterenol to cell cultures expressing mutant [Asn130]-beta ARs had no effect on intracellular levels of cAMP, indicating that the mutant beta AR is unable to affect stimulation of adenylate cyclase. These results indicate that substitution of the aspartic acid with asparagine at residue 130 of the human beta AR dissociates the well-characterized guanine nucleotide effects on agonist affinity from those on activation of the stimulatory guanine nucleotide-binding regulatory protein and adenylate cyclase and suggests the existence of two distinct counterions for the amine portion of catecholamines that are associated with high- and low-affinity agonist binding states of beta AR.

Effect of cholera toxin on histamine release from bone marrow-derived mouse mast cells.

Bone marrow-derived mouse mast cells were sensitized with monoclonal mouse IgE antibody and treated with cholera toxin (CT), which ADP-ribosylated the alpha-subunit of the stimulatory guanine nucleotide-binding regulatory protein Gs, prior to challenge with either antigen or thrombin. The CT treatment increased intracellular cAMP levels, but neither enhanced nor inhibited antigen-induced histamine release or arachidonate release. The same treatment of the sensitized bone marrow-derived mouse mast cells with CT markedly enhanced thrombin-induced histamine release without affecting arachidonate release. The CT treatment failed to affect antigen-induced and thrombin-induced generation of inositol trisphosphate and of diacylglycerol or mobilization of intracellular Ca2+. The results indicate that Gs in bone marrow-derived mouse mast cells is not involved in the transduction of the antigen-induced or thrombin-induced triggering signal to phospholipase C, which initiates the enhancement of phosphatidylinositol turnover. The enhancement of thrombin-induced histamine release by CT treatment with the observations that thrombin-induced histamine release was inhibited by pretreatment of the cells with pertussis toxin suggest that the involvement of a guanine nucleotide-binding regulatory protein in thrombin-induced biochemical events is an event distal to Ca2+ mobilization.