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Urotensin-II (U-II) is a vasoactive 'somatostatin-like' cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.

Calcitonin gene-related peptide (CGRP) is a neuropeptide with diverse biological properties including potent vasodilating activity. Recently, we reported the cloning of complementary DNAs (cDNAs) encoding the human and porcine CGRP receptors which share significant amino acid sequence homology with the human calcitonin receptor, a member of the recently described novel subfamily of G-protein-coupled 7TM receptors. Activation of this family of receptors has been shown to result in an increase in intracellular cAMP accumulation and calcium release. In this study, we demonstrate that HEK-293 cells expressing recombinant CGRP receptors (HEK-293HR or PR) respond to CGRP with increased intracellular calcium release (EC50 = 1.6 nM) in addition to the activation of adenylyl cyclase (EC50 = 1.4 nM). The effect of CGRP on adenylyl cyclase activation and calcium release was inhibited by CGRP (8-37), a CGRP receptor antagonist. Both effects were mediated by cholera toxin-sensitive G-proteins, but these two signal transduction pathways were independent of each other. While cholera toxin pretreatment of HEK-293PR cells resulted in permanent activation of adenylyl cyclase, the same pretreatment resulted in an inhibition of CGRP-mediated [Ca2+]i release. Pertussis toxin was without effect on CGRP-mediated responses. In addition, CGRP-mediated calcium release appears to be due to release from a thapsigargin-sensitive intracellular calcium pool. These results show that the recombinant human as well as porcine CGRP receptor can independently increase both cAMP production and intracellular calcium release when stably expressed in the HEK-293 cell line.

GAIP is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by some G protein alpha subunits. In the present studies, we have examined the structural basis for the ability of GAIP to discriminate among members of the Galphai family. Galphai1, Galphai3, and Galphao interacted strongly with GAIP, whereas Galphai2 interacted weakly and Galphas did not interact at all. A chimeric G protein composed of a Galphai2 N terminus and a Galphai1 C terminus interacted as strongly with GAIP as native Galphai1, whereas a chimeric N-terminal Galphai1 with a Galphai2 C terminus did not interact. These results suggest that the determinants responsible for GAIP selectivity between these two Galphais reside within the C-terminal GTPase domain of the G protein. To further localize residues contributing to G protein-GAIP selectivity, a panel of 15 site-directed Galphai1 and Galphai2 mutants were assayed. Of the Galphai1 mutants tested, only that containing a mutation at aspartate 229 located at the N terminus of Switch 3 did not interact with GAIP. Furthermore, the only Galphai2 variant that interacted strongly with GAIP contained a replacement of the corresponding Galphai2 Switch 3 residue (Ala230) with aspartate. To determine whether GAIP showed functional preferences for Galpha subunits that correlate with the binding data, the ability of GAIP to enhance the GTPase activity of purified alpha subunits was tested. GAIP catalyzed a 3-5-fold increase in the rate of GTP hydrolysis by Galphai1 and Galphai2(A230D) but no increase in the rate of Galphai2 and less than a 2-fold increase in the rate of Galphai1(D229A) under the same conditions. Thus, GAIP was able to discriminate between Galphai1 and Galphai2 in both binding and functional assays, and in both cases residue 229/230 played a critical role in selective recognition.

Clinical studies conducted with carvedilol suggest that beta-adrenoceptor antagonism is an effective therapeutic approach to the treatment of heart failure. However, many beta-adrenoceptor antagonists are weak partial agonists and possess significant intrinsic sympathomimetic activity (ISA), which may be problematic in the treatment of heart failure. In the present study, the ISAs of bucindolol, xamoterol, bisoprolol, and carvedilol were evaluated and compared in normal rats [Sprague-Dawley (SD)], in rats with confirmed heart failure [spontaneously hypertensive heart failure (SHHF)], and in isolated neonatal rat cardiomyocytes. At equieffective beta1-adrenolytic doses, the administration of xamoterol and bucindolol produced a prolonged, equieffective, and dose-related increase in heart rate in both pithed SD rats (ED50 = 5 and 40 microgram/kg, respectively) and SHHF rats (ED50 = 6 and 30 microgram/kg, respectively). The maximum effect of both compounds in SHHF rats was approximately 50% of that observed in SD rats. In contrast, carvedilol and bisoprolol had no significant effect on resting heart rate in the pithed SD or SHHF rat. The maximum increase in heart rate elicited by xamoterol and bucindolol was inhibited by treatment with propranolol, carvedilol, and betaxolol (beta1-adrenoceptor antagonist) but not by ICI 118551 (beta2-adrenoceptor antagonist) in neonatal rat. When the beta-adrenoceptor-mediated cAMP response was examined in cardiomyocytes, an identical partial agonist/antagonist response profile was observed for all compounds, demonstrating a strong correlation with the in vivo results. In contrast, GTP-sensitive ligand binding and tissue adenylate cyclase activity were not sensitive methods for detecting beta-adrenoceptor partial agonist activity in the heart. In summary, xamoterol and bucindolol, but not carvedilol and bisoprolol, exhibited direct beta1-adrenoceptor-mediated ISA in normal and heart failure rats.

The pharmaceutical industry has readily embraced genomics to provide it with new targets for drug discovery. Large scale DNA sequencing has allowed the identification of a plethora of DNA sequences distantly related to known G protein-coupled receptors (GPCRs), a superfamily of receptors that have a proven history of being excellent therapeutic targets. In most cases the extent of sequence homology is insufficient to assign these 'orphan' receptors to a particular receptor subfamily. Consequently, reverse molecular pharmacological and functional genomic strategies are being employed to identify the activating ligands of the cloned receptors. Briefly, the reverse molecular pharmacological methodology includes cloning and expression of orphan GPCRs in mammalian cells and screening these cells for a functional response to cognate or surrogate agonists present in biological extract preparations, peptide libraries, and complex compound collections. The functional genomics approach involves the use of 'humanized yeast cells, where the yeast GPCR transduction system is engineered to permit functional expression and coupling of human GPCRs to the endogenous signalling machinery. Both systems provide an excellent platform for identifying novel receptor ligands. Once activating ligands are identified they can be used as pharmacological tools to explore receptor function and relationship to disease.

Proliferation, differentiation and regulated trafficking of cells are the hallmarks of development and embryogenesis. This led us to speculate a role for chemokines and their receptors in this process. Here, we report the molecular cloning of AvCRL1, a novel member of the G-protein coupled receptor family from early stage 3 days old chick embryos. While the function and ligand for this receptor remain unknown, its sequence and gene structure indicates that it is most related to the family of chemokine receptors, with highest homology to the virally induced human BLR-1 and the CXCR3 or gammaIP-10/Mig-1 receptors.

The aggregation of activated platelets is mediated by the binding of fibrinogen to its cell surface receptor, the integrin alphaIIbbeta3. The recognition of fibrinogen by alphaIIbbeta3 depends, in part, on the tripeptide sequence Arg-Gly-Asp (RGD) in the adhesive protein. The interactions of a cyclic RGD-containing pentapeptide, [3H]-SK&F-107260, and a 1,4-benzodiazepine-based nonpeptide [3H]-SB-214857, with purified alphaIIbbeta3 have been investigated. Both compounds potently inhibit platelet aggregation at submicromolar concentrations. Binding of both [3H]-SK&F-107260 (Kd = 1.19 nM) and [3H]-SB-214857 (Kd = 1.85 nM) to alphaIIbbeta3 is of high affinity and fully reversible. The binding is monophasic, indicating a single class of noncooperative binding sites. The two radioligands exhibited similar values in binding to alphaIIbbeta3 purified on an RGD-affinity column (Bmax = 0.2 mol/mol alphaIIbbeta3) or to alphaIIbbeta3 purified over a lentil lectin column (Bmax = 0.03 mol/mol alphaIIbbeta3), suggesting that SK&F-107260 and SB-214857 interact with the same population of receptors. Binding of [3H]-SK&F-107260 and [3H]-SB-214857 to alphaIIbbeta3 require divalent cations, Mg++, Ca++ and Mn++ are able to support binding, with Mn++ being the most effective. Thirteen alphaIIbbeta3 antagonists, including four linear and three cyclic RGD peptides, five peptidomimetics, the fibrinogen gamma-chain dodecapeptide (HHLGGAKQAGDV) and the snake venom protein, echistatin, complete for [3H]-SK&F-107260 or [3H]-SB-214857 binding to alphaIIbbeta3. The affinity constants (Ki) of these compounds, determined by the two radioligand binding assays, are similar. Furthermore, these compounds exhibit the same rank order of potency in inhibiting biotinylated-fibrinogen binding to alphaIIbbeta3. Scatchard plot analyses of the [3H]-SK&F-107260 binding isotherms in the presence of unlabeled SB-214857 and gamma-chain dodecapeptide reveal competitive-type antagonism, indicating that SB-214857, gamma-chain dodecapeptide and SK&F-107260 interact with mutually exclusive binding sites on alphaIIbbeta3.

Chemokines play an important role in the regulation of endothelial cell (EC) function, including proliferation, migration and differentiation during angiogenesis, and re-endothelialization after injury. In this study, reverse transcriptase-polymerase chain reaction was used to reveal expression of various CXC and CC chemokine receptors in human umbilical vein EC. Northern analysis showed that CXCR4 was selectively expressed in vascular EC, but not in smooth muscle cells. Compared with other chemokines, stromal cell-derived factor-1alpha (SDF-1alpha), the known CXCR4 ligand, was an efficacious chemoattractant for EC, causing the migration of approximately 40% input cells with an EC50 of 10-20 nM. Of the chemokines tested, only SDF-1alpha induced a rapid, though variable mobilization of intracellular Ca2+ in EC. Experiments with actinomycin D demonstrated that CXCR4 transcripts were short-lived, indicating a rapid mRNA turnover. Interferon-gamma (IFN-gamma) caused a pronounced down-regulation of CXCR4 mRNA in a concentration- and time-dependent manner. In a striking functional correlation, IFN-gamma treatment also attenuated the chemotactic response of EC to SDF-1alpha. IL-1beta, tumor necrosis factor-alpha, and lipopolysaccharide produced a time course-dependent biphasic effect on CXCR4 transcription. Expression of CXCR4 in EC is significant, more so as it and several CC chemokine receptors have been shown to serve as fusion co-receptors along with CD4 during human immunodeficiency virus infection. Taken together, these findings provide evidence of chemokine receptor expression in EC and offer an explanation for the action of chemokines like SDF-1alpha on the vascular endothelium.

Access to DNA databases has introduced an exciting new dimension to the way biomedical research is conducted. 'Genomic research' offers tremendous opportunity for accelerating the identification of the cause of disease at the molecular level and thereby foster the discovery of more selective medicines to improve human health and longevity. The current challenge is to close the gap rapidly between gene identification and clinical development of efficacious therapeutics. In the present review, Jeffrey Stadel, Shelagh Wilson and Derk Bergsma outline the rationale and describe strategies for converting one large class of novel genes, orphan G protein-coupled receptors (GPCRs), into therapeutic targets. Historically, the superfamily of GPCRs has proven to be among the most successful drug targets and consequently these newly isolated orphan receptors have great potential for pioneer drug discovery.

The possibility that the cellular mechanism underlying adaptive supersensitivity in right and left atria of the guinea pig may involve either adenylyl cyclase or components of that transduction process was examined in left and right atria obtained from controls or guinea pigs chronically treated with reserpine. Adenylyl cyclase activity and the abundance of alpha-subunits of several G-proteins (i.e. Gs, Gi, and Go) were quantified using standard techniques. Functional concentrations of Gs and Gi were compared in tissues from control and treated animals using pertussis- or cholera toxin-induced protein ribosylation. Chronic treatment with reserpine did not alter basal levels of adenylyl cyclase activity in left or right atrium but did increase significantly the ability of isoproterenol, 5'-guanylylimido diphosphate, and forskolin to activate adenylyl cyclase in the left atrium compared with the control. In contrast, treatment with reserpine increased the ability of only isoproterenol to active adenylyl cyclase in the right atrium. The increases in enzyme activation were not correlated with any detectable change in the concentrations of G-proteins or beta-adrenoceptors. The correlation between the specificity of changes in responsiveness and increased activation of adenylyl cyclase suggests that the cellular mechanism that underlies the development of adaptive supersensitivity in the guinea pig myocardium may involve a modification of adenylyl cyclase. The data also support the idea that the development of enhanced responsiveness in cardiac muscle may not only involve more than one cellular mechanism but may even differ between right and left atrium and ventricles of the same species.

Osteopontin (OPN), an arginine-glycine-aspartate (RGD)-containing adhesive glycoprotein, is constitutively expressed in rat aorta and carotid arteries and is markedly elevated in response to vascular injury. OPN is chemotactic for vascular smooth muscle cells (SMCs), suggesting a role in vascular remodeling. However, the mechanism for the regulation of OPN expression is poorly understood. In the present study, the effect of platelet-derived growth factor (PDGF) on OPN mRNA expression was investigated in cultured rat aortic SMCs (RASMCs). When RASMCs were stimulated with 1 nmol/L PDGF, a 2.4-fold increase in OPN mRNA expression was observed at 3 hours (P < .05) that peaked at 14 hours with a 6.7-fold increase (P < .001). This induction was blocked by a monoclonal anti PDGF antibody. Further studies revealed that OPN mRNA expression was induced by PDGF-AB or PDGF-BB but not by PDGF-AA, indicating that only the beta-type PDGF receptor mediates this response. Compared with basic fibroblast growth factor, epidermal growth factor, transforming growth factor-beta, and interleukin-1 beta, PDGF was the most potent factor studied to induce OPN mRNA expression in RASMCs. Immunohistochemical studies demonstrated the elevation of OPN protein in PDGF-stimulated RASMCs. The temporal expression of OPN mRNA after rat carotid artery balloon angioplasty as assessed by both reverse transcription-polymerase chain reaction and Northern blot analysis revealed a 1.5-fold increase at 6 hours (P < .01) that peaked at 1 and 3 days with a 3.1-fold increase (P < .001). Immunohistochemical studies of carotid artery after angioplasty localized OPN expression in the medical SMCs at 1 day, ie. at a time of significant platelet adherence to the injured vessel, and thereafter to the intimal lesion during neointimal formation. These data suggest that OPN expression in vascular SMCs is regulated by PDGF through the beta-type PDGF receptor in vitro, and possibly in vivo in situations that involve PDGF released from platelets or other cellular sources, such as blood vessels after angioplasty injury.

CC-chemokine receptors form a closely linked locus in the p21-23 region on the short arm of human chromosome 3. We report the isolation of a novel chemokine receptor like gene from a 'human chromosome 3 specific' genomic library, screened by cross-hybridization with cDNA probes derived from known chemokine receptors. The gene, CKR-SKG2 contains an open reading frame that encodes a 359-377 amino acid protein, with two potential in frame start codons. However, comparative Southern blot analysis of human, mouse and hamster genomic DNA, revealed that CKR-SKG2 is the hamster ortholog of a novel chemokine receptor. This presumably occurred because chromosome 3 used for construction of the genomic library were purified from a human-hamster somatic cell hybrid. Further studies are underway to identify and clone the putative human homolog of this novel chemokine receptor.

The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily that mediates cell attachment on arginine-glycine-aspartic acid (RGD)-containing adhesive proteins. A solid-phase microtiter assay was developed to investigate the binding properties of purified alpha v beta 3, using tritiated [3H]SK&F-107260 as the radiolabeled ligand. alpha v beta 3, purified from human platelets, human placenta, and chicken osteoclasts, bound [3H]SK&F-107260 saturably and specifically. Saturation binding studies using platelet alpha v beta 3 revealed a single class of high affinity binding sites, exhibiting a Kd of 1.44 nM and Bmax of 0.20 mol of [3H]SK&F-107260/mol of alpha v beta 3. [3H]SK&F-107260 binding was inhibited by a variety of RGD-containing peptides and by the snake venom protein echistatin, whereas an RGE-containing peptide and four nonpeptide fibrinogen receptor (alpha IIb beta 3) antagonists failed to do so. This study shows that alpha v beta 3 exhibits distinct ligand specificity from the structurally homologous fibrinogen receptor, alpha IIb beta 3. The relative potencies of the RGD-containing peptides in inhibiting [3H]SK&F-107260 binding to alpha v beta 3 were the same as their relative potencies in inhibiting biotinylated-fibrinogen binding to the receptor. alpha v beta 3 purified from chicken osteoclasts and human placenta bound [3H]SK&F-107260 with similar affinities and displayed the same pharmacological profile as the platelet vitronectin receptor. The alpha v beta 3 antagonists inhibited the attachment of MG63 human osteosarcoma cells or rat osteoclasts to recombinant rat osteopontin. The rank order of potency of the antagonists in the cell adhesion assays was similar to that of the receptor binding assay, suggesting that the purified alpha v beta 3-[3H]SK&F-107260 binding assay is a valid reflection of the ligand binding to alpha v beta 3 on cell systems.

Calcitonin gene-related peptide (CGRP), a 37-amino-acid peptide, is a member of a small family of peptides including amylin or islet amyloid polypeptide and salmon calcitonin. These related peptides have been shown to display similar effects on in vitro and in vivo carbohydrate metabolism. The present study was initiated to identify and characterize the binding sites for these peptides in lung and nucleus accumbens membranes prepared from pig and guinea pig. Both tissues in either species displayed high-affinity (2-[125I]iodohistidyl10)humanCGRP alpha ([125I]hCGRP alpha) binding (IC50 = 0.4-7.7 nM), which was displaced by hCGRP8-37 alpha with equally high affinity (IC50 = 0.4-7.3 nM). High-affinity binding for [125I]Bolton-Hunter human amylin ([125I]BH-h-amylin) was also observed in these tissues (IC50 = 0.2-6.0 nM). In membranes from the nucleus accumbens of both species, salmon calcitonin competed for amylin binding sites with high affinity (IC50 = 0.1 nM) but was poor in competing for amylin binding in lung membranes. Rat amylin8-37 competed for [125I]hCGRP alpha binding with higher affinity (IC50 = 5.4 nM) compared with [125I]BH-h-amylin binding (IC50 = 200 nM) in porcine nucleus accumbens, whereas in guinea pig nucleus accumbens, the IC50 values for rat amylin8-37 were 117 and 12 nM against [125I]hCGRP alpha and [125I]BH-h-amylin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The potent vasoactive peptide hormone endothelin (ET) binds to receptors which belong to the G-protein coupled receptor family. The availability of non-peptide antagonists for ET receptors allows investigation of the relationship among the binding sites for peptide and non-peptide ligands. In this study, a lysine residue, conserved within transmembrane domain 3 (TM3) of the ETA and ETB receptor subtypes, is implicated in agonist and antagonist binding by its analogous position within TM3 to a binding site aspartate residue conserved within bioactive amine receptors. Replacement of this lysine within hETB by arginine, alanine, methionine, aspartate, or glutamate results in hETB variants with unaltered affinities for agonist peptide ET-1 but which have affinities for peptide agonists ET-2, ET-3, sarafotoxin 6C, and TRL 1736 which are between 1-3 orders of magnitude lower than their corresponding wild-type hETB values. Significantly, the affinities of non-peptide antagonists, (+/-)-SB 209670 and its analogs as well as Ro 46-2005, are abrogated. The results suggest that an interaction of K182 of hETB with the indan 2-carboxyl of (+/-)-SB 209670 may contribute to the high-affinity binding of the diarylindan antagonists. The results indicate that TM3 of hETB is a region of overlap among the binding sites of non-peptide antagonists and the affected peptide agonists.

The guanine nucleotide regulatory protein, GS, mediates transmembrane signaling by coupling membrane receptors to the stimulation of adenylyl cyclase activity. The full length coding sequences for the M(r) = 42-45,000, short form (S), and M(r) = 46-52,000, long form (L), of the alpha-subunits of rat GS were placed in yeast expression vectors under the regulatory control of the copper-inducible CUP1 promoter and transformed into Saccharomyces cerevisiae. In the presence of 100 microM CuSO4, the transformed yeast expressed GS-alpha mRNAs and proteins. In reconstitution experiments, rat GS-alpha(S and L), solubilized from yeast membranes with 1% cholate, conferred NaF-, (-)isoproterenol-, and guanine nucleotide-dependent sensitivity to adenylyl cyclase catalytic units in S49 lymphoma cyc- cell membranes, which are devoid of endogenous GS-alpha. GS-alpha (S) demonstrated twice the activity of GS-alpha(L) in reconstitution assays of fluoride-stimulated adenylyl cyclase activity. Comparison of GS-alpha (S) expressed in yeast with GS purified from rabbit liver or human erythrocytes showed that the crude recombinant protein was fully competent in reconstituting NaF-stimulated adenylyl cyclase activity, but was only 2-5% as potent as purified GS. Addition of bovine brain beta gamma subunits during reconstitution enhanced all parameters of adenylyl cyclase activity for GS-alpha(S and L) obtained from yeast. In contrast, transducin beta gamma only enhanced agonist-stimulated adenylyl cyclase activity for GS-alpha (S and L) following reconstitution. These results demonstrate that the expression of functional mammalian GS-alpha subunits in yeast may be useful for their biochemical characterization.

Osteopontin (OPN), a 41-kDa phosphorylated glycoprotein, has been detected in rat aorta and carotid arteries, and expression of its mRNA in blood vessels is strongly increased in response to vascular injury. To investigate the potential role of OPN in vascular pathophysiology, we studied the effect of rat OPN on aortic smooth muscle cell migration and proliferation in vitro. OPN enhanced the migration of rat smooth muscle cells in a time- and concentration-dependent manner with an EC50 value of 46 +/- 11 nmol/liter (n = 5). The maximal increase in cell migration by OPN was 29-fold over basal levels. OPN-induced smooth muscle cell migration was inhibited in a concentration-dependent manner by the monoclonal antibody F11, which recognizes the rat integrin subunit beta 3. In contrast, polyclonal antiserum recognizing the rat integrin beta 1 subunit did not inhibit smooth muscle cell migration in response to OPN, but did block fibronectin-promoted migration. Moreover, OPN-induced smooth muscle cell migration was dependent on the presence of extracellular divalent cations and was significantly inhibited by anti-OPN antibodies. OPN did not stimulate [3H]thymidine incorporation into cultured smooth muscle cells, indicating that it selectively enhanced migration. In view of the pathological significance of arterial smooth muscle cell migration in the formation of intimal thickening, our results suggest that smooth muscle cell recognition of OPN, probably through the vitronectin receptor, alpha v beta 3, could play a role in the cells' response to vascular injury and especially neointima formation.

Thrombin stimulates cytosolic calcium mobilization and tritiated thymidine incorporation in rat glomerular mesangial cells. This effect may be mediated by a thrombin receptor similar to the receptor found in human platelets. In order to test this possibility, a series of analogues of the thrombin receptor peptide, SFLL-RNPNDKYEPF, was evaluated for their effects on mesangial cells. Analogues of the thrombin receptor peptide containing five, six, seven and 14 amino acids were as efficacious as thrombin with respect to calcium mobilization and thymidine incorporation, although they were significantly less potent. The dissimilarity in potency between thrombin and the thrombin receptor peptides is consistent with the kinetics of the proposed mechanism of action of the enzyme, since the cleavage by thrombin of its receptor results in a tethered ligand which is at a relatively high concentration compared to the free peptides in solution. Those thrombin receptor peptide analogues which showed decreased activity in platelets were tested in mesangial cells. Removal of serine at position one, N-acetylation, or replacement of the phenylalanine at position two with alanine resulted in analogues which were inactive in stimulating mesangial cell proliferation or calcium mobilization. In addition, those analogues which had no stimulatory effects in mesangial cells were not antagonists of SFLLRN-mediated calcium mobilization and thymidine incorporation in mesangial cells.

Molecular modeling and protein engineering techniques have been used to study residues within G-protein-coupled receptors that are potentially important to ligand binding and selectivity. In this study, Tyr-129 located in transmembrane domain 2 of the human endothelin (ET) type A receptor A (hETA) was targeted on the basis of differences between the hETA and type B receptor (hETB) sequences and the position of the residue on ET receptor models built using the coordinates of bacteriorhodopsin. Replacement of Tyr-129 of hETA by alanine, glutamine, asparagine, histidine, lysine, serine, or phenylalanine results in receptor variants with enhanced ET-3 and sarafotoxin 6C affinities but with unchanged ET-1 and ET-2 affinities. Except for Tyr-129-->Phe hETA, these hETA variants have two to three orders of magnitude lower binding affinity for the ETA-selective antagonist BQ123. Replacement of His-150, the residue in hETB that is analogous in sequence to Tyr-129 of hETA, by either tyrosine or alanine does not affect the affinity of peptide ligands. These results indicate that although transmembrane domain 2 is important in ligand selectivity for hETA, it does not play a significant role in the lack of ligand selectivity shown by hETB. Chimeric receptors have been constructed that further support these conclusions and indicate that at least two hETA regions contribute to ligand selectivity. Additionally, the data support an overlap in the binding site in hETA of agonists ET-3 and sarafotoxin 6C with that of the antagonist BQ123.

A secreted form of phospholipase A2 (PLA2) has been implicated in inflammatory disorders such as rheumatoid arthritis and sepsis. To determine if PLA2 may also play a role in allergic rhinitis, we have measured enzymic activity in nasal lavage from allergic subjects. Enhanced activity of PLA2 in the lavage was observed following nasal challenge with antigen or histamine. The PLA2 in the nasal lavage was partially purified by acid extraction, size exclusion chromatography, and ion exchange chromatography. The partially purified enzyme from nasal lavage was subsequently compared to a recombinant form of human PLA2 identified in synovial fluid from arthritic patients. The two enzymes showed similar molecular weights (15 to 16 kD) on SDS-PAGE, and both reacted with a rabbit polyclonal antiserum raised to a galactokinase-PLA2 fusion protein. The enzymatic activities of the two PLA2s were indistinguishable when compared for ionic dependence, substrate selectivity, and sensitivity to inhibitors. These results suggest that the PLA2 induced in nasal lavage in response to challenge by antigen is very similar to the extracellular PLA2 found in synovial fluid from subjects with rheumatoid arthritis and may play a role in the inflammatory processes associated with allergic rhinitis.

According to current models, human thrombin receptors are activated when thrombin cleaves the receptor's N terminus, exposing the tethered ligand domain, SFLLRN. In the megakaryoblastic CHRF-288 cell line, thrombin receptor activation is followed by the rapid internalization of > 90% of the receptors. In the present studies, antibodies directed at the site of cleavage by thrombin were used to examine changes in receptor structure during activation, internalization, and recovery. As would be expected, the initial rate of receptor cleavage was directly related to the thrombin concentration. However, even after prolonged incubation, receptor cleavage was incomplete until the thrombin concentration exceeded the receptor concentration. Only cleaved receptors were internalized in response to thrombin and only catalytically active thrombin and active variants of SFLLRN-containing peptides caused receptor internalization. Over a 3-h period following receptor activation by thrombin, there was a gradual recovery of approximately one-quarter of the receptors on the cell surface. These receptors were detectable with antibodies directed at retained portions of the receptor N terminus, but not with antibodies directed at the proposed site of cleavage, confirming that they are recycled, rather than new, receptors. At 4 degrees C two-thirds of the receptors cleaved by thrombin were retained on the cell surface. Like recycled receptors these "cold-cleaved" receptors failed to self-activate when warmed to 37 degrees C, but could be activated by SFLLRN. Unlike recycled receptors, however, the cold-cleaved receptors were also internalized and appeared to be activated by a second addition of thrombin. These results 1) provide strong evidence at the protein level that thrombin cleaves its receptors at the predicted site, 2) show that receptor activation is necessary for internalization, 3) suggest that each thrombin molecule may not activate large numbers of receptors, 4) demonstrate that a substantial fraction of internalized thrombin receptors can be recycled, and 5) suggest that the failure of recycled receptors to be reactivated by thrombin may involve a change in the receptor that does not occur at 4 degrees C. Finally, the inability of cold-cleaved receptors to self activate in the absence of thrombin, suggests that in addition to cleaving the receptor, thrombin may also play an important role in guiding the tethered ligand domain to regions on the remainder of the receptor that mediate activation.

This study details the investigation of induction of retractile shape change in the osteoclast through inhibition of adhesion between osteoclasts and matrix with (1) peptide analogs bearing an Arg-Gly-Asp (RGD) sequence, (2) antibodies to the integrin alpha V beta 3 vitronectin receptor, and (3) the RGD-containing snake venom peptide echistatin. Osteoclast retraction on dentin has been demonstrated for GRGDSP peptide, in contrast to the inactivity of the analog containing the conservative RGE sequence modification. An osteoclast adhesion assay employing rat or chick bone cells and serum-coated glass coverslips as substrate was developed for routine evaluation of inhibition of adhesion. Antibodies F4 and F11 to the beta 3 chain of rat vitronectin receptor were effective at submicromolar concentrations in rat osteoclasts (IC50 0.29 and 0.05 microM, respectively), whereas MAb 23C6 to human/chick vitronectin receptor was somewhat less effective against chick osteoclasts (IC50 1.6 microM). A rank order of RGD analog activity (mean IC50, microM) in the serum-coated glass adhesion assay was derived for the linear peptides GRGDSP (201 microM), GRGDTP (180 microM), Ac-RGDS-NH2 (84 microM), Ac-RGDV-NH2 (68 microM), RGDV (43 microM), GRGDS (38 microM), and RGDS (26 microM). The two most potent short peptides were the cyclic analog SK&F 106760 Ac-S,S-cyclo-(Cys-(N alpha Me)Arg-Gly-Asp-Pen)-NH2 (IC50 7.0 microM), and the Telios peptide H-Gly-S,S-cyclo-(Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys)-Ala-OH (IC50 6.6 microM). The snake venom peptide echistatin was the most potent substance evaluated in the serum-coated glass assay (IC50 0.78 nM) employing either rat or chick osteoclasts.(ABSTRACT TRUNCATED AT 250 WORDS)

A secreted form of phospholipase A2 (PLA2) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA2 was cloned from a human placental cDNA library. The cDNA encoding the human PLA2 was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca 1 mg/L of recombinant PLA2 into the medium, was scaled up in culture to 180 L. The recombinant enzyme was purified from the cell supernatant to apparent homogeneity by a novel procedure combining adsorption to poly(vinylidene difluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLA2 activity was 58%. A direct comparison between the purified recombinant human PLA2 and PLA2 purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLA2 inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA2 can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA2 inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLA2 in inflammatory disease.

Chronic treatment with reserpine (0.1 mg/kg/day x 7 days) leads to the development of adaptive supersensitivity of ventricular myocardium of guinea pigs. The compensatory increase in sensitivity is associated with a small increase in beta-adrenoreceptor number. However, sensitivity is increased to a number of agonists that do not interact with beta-adrenoceptors. An evaluation of the role of both adenylyl cyclase and guanine nucleotide-binding regulatory proteins in the development of adaptive supersensitivity was carried out using crude membrane fragments from untreated control and chronically reserpine-treated guinea pigs. Quantitative analysis of Gs alpha and Gi protein concentrations was accomplished using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Chronic treatment with reserpine reduced basal levels of adenylyl cyclase activity by nearly 60%. The reduced activity was not the result of a loss of endogenous norepinephrine, because incubation of tissues in the presence of propranolol did not alter the basal level of adenylyl cyclase activity. Incubation in the presence of guanylylimido diphosphate (10(-5) M) also significantly reduced basal adenylyl cyclase activity, by nearly 70%. Chronic treatment with reserpine failed to significantly alter the activation of adenylyl cyclase by isoproterenol, impromidine, NaF, or forskolin. These data suggest that chronic treatment with reserpine does not alter agonist-induced activation of adenylyl cyclase. Furthermore, analysis of Gs alpha and Gi indicated that chronic treatment with reserpine did not affect the levels of these regulatory proteins in ventricular myocardial membranes. The data indicate that the enhanced sensitivity of guinea pig ventricular myocardium is not the result of an alteration in adenylyl cyclase activity or in the concentration of guanine nucleotide regulatory proteins. Therefore, the enhanced responsiveness to widely diverse agonists must be due to an alteration in cellular function beyond the level of adenylyl cyclase.

A human liver cDNA library was screened using a rat type 1 angiotensin II receptor cDNA coding sequence as a probe. cDNA clones were isolated which encoded a protein of 359 amino acids that shared 94.4% and 95.3% identify to rat and bovine type 1 angiotensin II receptors, respectively. Ligand binding studies of the cloned receptor expressed in COS cells suggested that it is pharmacologically a type 1 angiotensin II receptor subtype. Electrophysiological studies of the receptor expressed in Xenopus laevis oocytes revealed that it could functionally couple to a second messenger system leading to the mobilization of intracellular stores of calcium. Southern and Northern blot analyses indicated that the cloned receptor is represented as a single copy in the human genome and is expressed in many tissues of different histogenic origin with the exception of brain, where mRNA transcripts were barely detectable.

The role of the alpha subunit of the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase (GS alpha) in the down-regulation of beta-adrenergic receptors by pindolol was studied in S49 cyc- cells (normally GS alpha-deficient) transfected to express functional recombinant rat GS alpha. An inducible cell line (S49 GS alpha IND) was derived from S49 cyc- cells transfected with a vector containing the full-length coding sequence of GS alpha under the inducible control of the mouse mammary tumor virus long-terminal repeat promoter. GS alpha was not detectable in S49 GS alpha IND cells by immunoblot or by ADP-ribosylation in the presence of cholera toxin and [alpha-32P]NAD. When cells were grown in 100 nM dexamethasone, isoproterenol-stimulated cyclic AMP accumulation increased within 3 h. After 15 h, GS alpha was present at a level 40-50% of that found in S49 wild-type (WT) cells as measured either by immunoblot analysis or by [alpha-32P]ADP-ribosylation. Membranes prepared from GS alpha IND cells grown in the presence of dexamethasone bound agonist with high affinity, and this binding was sensitive to guanine nucleotides. A second vector, DzbGS alpha +, contained the coding sequence of GS alpha under the constitutive regulatory control of the SV40 early promoter. This vector was introduced into cyc- cells, and the resulting cells, S49 GS alpha CST cells, expressed GS alpha at a level comparable to that found in S49 WT cells as measured by immunoblot analysis. Isoproterenol-stimulated cyclic AMP accumulation in S49 GS alpha CST cells was at least as great as in S49 WT cells. When cells were grown in the presence of dexamethasone, exposure to 50 nM pindolol for 12 h down-regulated the density of beta-adrenergic receptors in S49 WT cells to 60% of that in cells grown in the absence of pindolol, but pindolol had no effect on the density of receptors on cyc- or GS alpha IND cells. When GS alpha CST cells were exposed to 50 nM pindolol for 12 h, the density of beta-adrenergic receptors was down-regulated by the same amount as in S49 WT cells. These results suggest that GS alpha is necessary to restore the ability of pindolol to down-regulate beta-adrenergic receptors in S49 cyc- cells and that the protein must be expressed at a level comparable to that found in S49 WT cells.

Platelet-activating factor (PAF) is an unusually potent phospholipid known to be produced by neuronal cells and to modulate cerebral blood flow and metabolism. In previous studies with NCB-20 cells, we reported that PAF induced a significant mobilization of intracellular free Ca2+ ([Ca2+]i), which was inhibited by PAF antagonists. The increase was the result of release from intracellular stores and influx from extracellular sources. The present study was designed to characterize further PAF receptor-mediated cellular signal-transduction mechanisms in myo-[3H]inositol-labeled cells. PAF induced a concentration-dependent increase in phosphatidylinositol (Pl) metabolism, with EC50 values of 1.96 +/- 0.62 nM and 1.12 +/- 0.50 nM for inositol trisphosphate (IP3) and inositol monophosphate (IP1) formation, respectively (four experiments). The maximal production of IP3 and IP1 induced by 50 nM PAF was 254 +/- 34% and 178 +/- 25% over the basal, respectively (four experiments). PAF-induced Pl metabolism was concentration-dependently inhibited by the PAF antagonist BN50739, with an IC50 value of 6.48 +/- 0.52 nM (four experiments). The protein kinase C (PKC) activator phorbol 12,13-dibutyrate concentration-dependently inhibited PAF-induced Pl metabolism and [Ca2+]i mobilization in NCB-20 cells, of NCB-20 cells with pertussis toxin (PTX) resulted in a concentration-dependent inhibition of PAF-induced IP3 production and intracellular Ca2+ release, with a maximal reduction of 66.9 +/- 3.5% and 63 +/- 6.1%, respectively, at 300 ng/ml PTX. PTX in the presence of [32P]NAD specifically [32P]ADP-ribosylated a 38-kDa protein in membranes prepared from NCB-20 cells. Pretreatment of the cells with PTX resulted in a concentration-dependent inhibition of subsequent 32P-labeling of the toxin substrate in the membranes and correlated with the uncoupling of PAF-induced IP3 formation. PAF (0.01-10 nM) elicited a concentration-related stimulation in guanosine 5'-O-(3-[35S]) triphosphate ([35S]GTP gamma S) binding to G alpha i(1,2) proteins, which was inhibited by the PAF antagonist BN50739. PAF at 10 nM also increased [35S]GTP gamma S binding to G alpha s and G alpha o. PAF-evoked activation of G alpha i(1,2) and G alpha o was reduced by preincubation with PTX. Our results reveal that neuronal cells possess PAF receptors linked through guanine nucleotide-binding proteins to phospholipase C and receptor-operated Ca2+ channels that are regulated by PKC. Both PTX-sensitive and -insensitive guanine nucleotide-binding proteins appear to couple the PAF receptor to activation of phospholipase C and the increase in [Ca2+]i. These results contribute to the further understanding of the mechanisms behind PAF actions on neuronal cells.

Studies by various investigators have demonstrated that the low Km, cAMP-specific phosphodiesterase (PDE IV) is selectively inhibited by a group of compounds typified by rolipram and Ro 20-1724. In addition to inhibiting the catalytic activity of PDE IV, rolipram binds to a high affinity binding site present in brain homogenates. Although it has been assumed that the high affinity rolipram-binding site is PDE IV, no direct evidence has been produced to support this assumption. The present studies were undertaken to determine whether the rolipram-binding site is coexpressed with PDE IV catalytic activity in Saccharomyces cerevisiae genetically engineered to express human recombinant monocytic PDE IV (hPDE IV). Expressing hPDE IV cDNA in yeast resulted in a 20-fold increase in PDE activity that was evident within 1 h of induction and reached a maximum by 3-6 h. The recombinant protein represented hPDE IV as judged by its immunoreactivity, molecular mass (approximately 88 kDa), kinetic characteristics (cAMP Km = 3.1 microM; cGMP Km greater than 100 microM), sensitivity to rolipram (Ki = 0.06 microM), and insensitivity to siguazodan (PDE III inhibitor) and zaprinast (PDE V inhibitor). Saturable, high affinity [3H] (R)-rolipram-binding sites (Kd = 1.0 nM) were coexpressed with PDE activity, indicating that both binding activity and catalytic activity are properties of the same protein. A limited number of compounds were tested for their ability to inhibit hPDE IV catalytic activity and compete for [3H](R)-rolipram binding. Analysis of the data revealed little correlation (r2 = 0.35) in the structure-activity relationships for hPDE IV inhibition versus competition for [3H] (R)-rolipram binding. In fact, certain compounds (e.g. (R)-rolipram Ro 20-1724) possessed a 10-100-fold selectivity for inhibition of [3H] (R)-rolipram binding over hPDE IV inhibition, whereas others (e.g. dipyridamole, trequinsin) possessed a 10-fold selectivity for PDE inhibition. Thus, although the results of these studies demonstrate that hPDE IV activity and high affinity [3H](R)-rolipram binding are properties of the same protein, they do not provide clear cut evidence linking the binding site with the PDE inhibitory activity of rolipram and related compounds.

The alpha subunit of the guanine nucleotide-binding regulatory protein GS mediates stimulation of adenylyl cyclase activity. This subunit, GS alpha, exists as two molecular weight forms, termed long and short, that differ by 14 or 15 amino acids. A physiological distinction between these two forms has yet to be defined. To compare the activities of these GS alpha isoforms, long and short forms of rat GS alpha were expressed in the cyc- variant of S49 murine lymphoma cells, which is deficient in endogenous GS alpha expression. By immunoblot analysis, the level of recombinant proteins in the clones expressing the long form of GS alpha was about twice that present in the clones expressing the short form of GS alpha or in the S49 wild-type cells. Both recombinant GS alpha proteins were sensitive to cholera toxin-catalyzed ADP-ribosylation, although the short form was labeled preferentially in both recombinant and S49 wild-type cell lines. In whole-cell assays, the clones expressing the long and short forms of GS alpha and the S49 wild-type cells gave comparable responses for stimulation of cAMP accumulation after challenge with (-)-isoproterenol, cholera toxin, or forskolin. In adenylyl cyclase assays with partially purified membranes, clones expressing the long form of GS alpha gave approximately twice the levels of cAMP in response to isoproterenol, guanosine-5'-O-(3-thio)triphosphate, NaF, or forskolin, compared with membranes from the clones expressing the short form of GS alpha or the S49 wild-type cells. However, when maximal adenylyl cyclase activity was normalized to the level of GS alpha protein in S49 wild-type cells, the cAMP productions were similar between all of the cell lines. In other membrane-based assays, the long and short forms of GS alpha were also equivalent in their dose response to isoproterenol and GTP, their kinetics of guanine nucleotide exchange and GTPase activity, and the induced high and low affinity states of the beta-adrenergic receptor in response to isoproterenol. In the latter radioligand binding analysis, membranes from the two clones expressing the long form of GS alpha consistently gave a greater proportion of the agonist high affinity state; however, this variation likely reflects the greater expression levels of GS alpha in these membranes. Thus, we conclude that the long and short forms of GS alpha expressed in S49 cyc- cells are very similar in their ability to stimulate adenylyl cyclase activity and to couple to beta-adrenergic receptors.

Changes in the intracellular concentration of calcium [( Ca2+]i) have been shown to mediate the physiological effects of certain agonists. Ca2+ mobilization occurs through multiple mechanisms which involve both influx and internal release of Ca2+. Prostaglandin F2 alpha (PGF2 alpha) caused a transient mobilization of intracellular Ca2+ in 3T3-L1 fibroblasts. This effect was characterized by fluorescence measurements of trypsin-treated cells loaded with fura-2/AM. In the absence of extracellular Ca2+, the peak amount of Ca2+ mobilized by PGF2 alpha was decreased by 70%, a lag time before the onset of [Ca2+]i increase was observed, and the rate of rise of [Ca2+]i was slowed. Addition of NaF (10 mM) to fura-2-loaded 3T3-L1 cells caused a dose-dependent increase in [Ca2+]i after a brief (approximately 10 s) lag. Maximal effects (approximately 300 nM) were observed at 5-10 mM-NaF. This effect was dependent on the presence of extracellular Ca2+ and appeared to be independent of inositol phosphate production. After reaching a peak at around 40 s after fluoride addition, [Ca2+]i returned to near-baseline within 120 s. This return of [Ca2+]i to near-baseline after fluoride stimulation and the inability of the cells to respond to a subsequent addition of fluoride indicated that the response to fluoride underwent desensitization. Similarly, the pathway used by PGF2 alpha to mobilize Ca2+ underwent desensitization. Exposure of the cells to a maximally effective concentration of fluoride and subsequent addition of PGF2 alpha produced a [Ca2+]i response to PGF2 alpha which was similar in magnitude and kinetics to that seen for PGF2 alpha in the absence of extracellular Ca2+. Conversely, prior exposure of cells to PGF2 alpha diminished the ability of fluoride to mobilize Ca2+. PGF2 alpha also increased inositol phosphate formation, with a time course and dose-response consistent with its ability to increase [Ca2+]i. Prior exposure of cells to fluoride did not change the time course or dose-response characteristics of PGF2 alpha-induced generation of inositol phosphates. These data suggest that PGF2 alpha and fluoride share a common mechanism of activating Ca2+ influx in 3T3-L1 cells.

The guanine nucleotide binding protein (G-protein) dependency of several of the activities of tumor necrosis factor (TNF), including cytotoxicity, inhibition of lipoprotein lipase activity, blockade of 3T3-L1 differentiation, and receptor binding were examined. TNF induced killing of the TNF-sensitive cell line L929S (ED50 = 30 pM), but had little to no effect on the TNF-resistant cell line L929R (ED50 = 5,300 pM). TNF-induced cytotoxicity in L929S was antagonized in a dose-dependent manner by pertussis toxin (sevenfold increase in ED50). However, TNF-induced cytotoxicity in L929R cells was only minimally affected by pretreatment with a high dose (50 ng/ml) of pertussis toxin (1.5-fold increase in ED50). Parallel biochemical investigations revealed that inhibition was accompanied by toxin-induced ADP ribosylation of a Gi alpha-like subunit in L929 and 3T3-L1 cell membranes. Pertussis toxin also significantly reduced TNF-induced inhibition of lipoprotein lipase activity in 3T3-L1 adipocytes and TNF blockade of 3T3-L1 preadipocyte differentiation. However, pertussis toxin pretreatment of L929S, L929R, and 3T3-L1 cell cultures had little to no effect on TNF receptor binding. These data indicate that several TNF-induced biological activities in the L929 and 3T3-L1 cell lines are partially dependent upon a pertussis toxin-sensitive G-protein.

Scg1, the product of the Saccharomyces cerevisiae SCG1 (also called GPA1) gene, is homologous to the alpha subunits of G proteins involved in signal transduction in mammalian cells. Scg1 negatively controls the pheromone response pathway in haploid cells. Either pheromonal activation or an scg1 null mutation relieves the negative control and leads to an arrest of cell growth in the G1 phase of the cell cycle. Expression of rat G alpha s was previously shown to complement the growth defect of scg1 null mutants while not allowing mating. We have extended this analysis to examine the effects of the short form of G alpha s (which lacks 15 amino acids present in the long form), G alpha i2, G alpha o, and Scg1-mammalian G alpha hybrids. In addition, we have found that constructs able to complement scg1 are also able to inhibit the response to pheromone and mating when expressed in a wild-type SCG1 strain. Overexpression of Scg1 has a similar inhibitory effect. These results are consistent with a model proposed for the action of Scg1 as the alpha component of a heterotrimeric G protein in which the beta gamma component (Ste4/Ste18) activates the pheromone response after dissociation from Scg1. They suggest that the G alpha constructs able to complement scg1 can interact with beta gamma to prevent activation of the pathway but are unable to interact with pheromone receptors to activate the pathway.

To study the epigenetic regulation of beta adrenergic receptor subtypes, we examined the effects of phorbol esters on beta adrenergic receptor coupling to adenylyl cyclase in 3T3-L1 fibroblasts, which express both beta-1 and beta-2 adrenergic receptor subtypes. Pretreatment of intact 3T3-L1 cells with the protein kinase C activator phorbol dibutyrate caused a dose- and time-dependent decrease in subsequent cyclic AMP (cAMP) accumulation mediated by the beta adrenergic agonist isoproterenol. This effect was selective for beta-adrenergic receptor-mediated responses because there was a potentiation of cAMP accumulation caused by other activators such as prostaglandin E1, forskolin or cholera toxin. The inactive phorbol, alpha-phorbol dibutyrate was ineffective at 1 microM in attenuating isoproterenol stimulation, and 25 nM of the protein kinase C inhibitor staurosporine blocked the effects of phorbol ester on beta adrenergic agonist responses. Stimulation of cAMP accumulation by isoproterenol occurred through a greater proportion of beta-2 adrenergic receptors in phorbol dibutyrate-treated cells than in control cells. This was demonstrated using the beta-1 adrenergic selective antagonist ICI 89.406 and the beta-2 adrenergic selective antagonist ICI 118.551 to inhibit competitively isoproterenol-stimulated cAMP accumulation. Beta-2 adrenergic receptor number and subtype in these cells are regulated by glucocorticoids and butyrate. Decreasing the proportion of beta-1 adrenergic receptors and concomitantly increasing beta-2 adrenergic receptors with either glucocorticoids or butyrate decreased the ability of phorbol ester pretreatment to attenuate cAMP accumulation by isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)

The unique properties of agonist binding to the frog erythrocyte beta-adrenergic receptor include the existence of two affinity forms of the receptor. The proportion and relative affinity of these two states of the receptor for ligands varies with the intrinsic activity of the agonist and the presence of guanine nucleotides. The simplest model for hormone-receptor interactions which can explain and reproduce the experimental data involves the interaction of the receptor R with an additional membrane component X, leading to the agonist-promoted formation of a high affinity ternary complex HRX. Computer modeling of agonist binding data with a ternary complex model indicates that the model can fit the data with high accuracy under conditions where the ligand used is either a full or a partial agonist and where the system is altered by the addition of guanine nucleotide or after treatment with group-specific reagents, e.g. p-hydroxymercuribenzoate. The parameter estimates obtained indicate that the intrinsic activity of the agonist is correlated significantly with the affinity constant L of the component X for the binary complex HR. The major effect of adding guanine nucleotides is to destabilize the ternary complex HRX from which both the hormone H and the component X can dissociate. The modulatory role of nucleotides on the affinity of agonists for the receptor is consistent with the assumption that the component X is the guanine nucleotide binding site. The ternary complex model was also applied successfully to the turkey erythrocyte receptor system. The model provides a general scheme for the activation by agonists of adenylate cyclase-coupled receptor systems and also of other systems where the effector might be different.

The beta 2-adrenergic receptor from mouse 3T3-L1 cells is up-regulated through genetic mechanisms by glucocorticoids and butyrate. To study the genetic regulation of these receptors, we sequenced a 5 kb region of genomic DNA from 3T3-L1 cells, containing the beta-adrenergic receptor gene and approx. 1.5 kb of both 5' and 3' flanking sequences. The sequence contained one copy of an 8 bp consensus sequence which can confer phorbol ester-responsiveness to genes. Phorbol esters attenuated the up-regulation of beta 2-adrenergic receptors by glucocorticoids but not by butyrate. This effect was probably due to a phorbol ester-induced decrease in glucocorticoid receptor number. Using methylation-sensitive restriction enzymes, we examined the methylation of a CG-rich region occurring 5' to the gene and did not detect any changes in methylation of this region upon dexamethasone or butyrate treatment. A total of 16 putative glucocorticoid response elements were found which may mediate the glucocorticoid-induced increase in beta 2-adrenergic receptors. A comparison of the regulatory sequences of the two beta-adrenergic receptor subtypes from human and mouse confirms the observed physiological controls of receptor subtype expression and offers an explanation as to why the subtypes differ in genetic regulation.

Desensitization of turkey erythrocyte adenylate cyclase by exposure of these cells to the beta-adrenergic agonist isoproterenol leads to a decrease in subsequent adenylate cyclase stimulation by isoproterenol, F-, or Gpp(NH)p without any apparent loss or down regulation of receptors (B.B. Hoffman et al. J. Cyclic Nucl. Res. 5: 363-366, 1979). We now report that the desensitization is associated with a functional "uncoupling" of the beta-adrenergic receptor. This is evidenced by an impaired ability of receptors to form a high affinity, guanine nucleotide sensitive complex with agonist as assessed by computer analysis of radioligand binding data. The changes in adenylate cyclase responsiveness as well as the alterations in receptor affinity for agonists are reproduced by incubation of turkey erythrocytes with the cAMP analog 8-Bromo-adenosine 3':5'- cyclic monophosphate. These findings suggest that one possible mechanism for the development of desensitization in adenylate cyclase systems may be a cAMP mediated alteration of a component(s) of the beta-adrenergic receptor-adenylate cyclase complex which results in impaired receptor-cyclase coupling.

The guanine nucleotide regulatory protein component (N) of the frog erythrocyte membrane adenylate cyclase system appears to form a stable complex with the beta-adrenergic receptor (R) in the presence of agonist (H). This agonist-promoted ternary complex HRN can be solubilized with Lubrol. The guanine nucleotide regulatory protein associated with the solubilized complex can be adsorbed either to GTP-Sepharose directly or to wheat germ lectin-Sepharose via its interaction with the receptor which is a glycoprotein. Guanosine 5'-O-(3-thiotriphosphate)(GTP gamma S) can be used to elute the guanine nucleotide regulatory protein from either Sepharose derivative. The resulting N.GTP gamma S complex conveys nucleotide-dependent adenylate cyclase activity when combined with a Lubrol-solubilized extract of turkey erythrocyte membranes. The ability to observe GTP gamma S-dependent reconstitution of adenylate cyclase activity in the eluate from either resin required the formation of the HRN complex prior to solubilization. The N protein can be identified by its specific [32P]ADP ribosylation catalyzed by cholera toxin in the presence of [32P]NAD+. The existence of a stable HRN intermediate complex is supported by the observation that agonist pretreatment of frog erythrocyte membranes results in a 100% increase in the amount of 32P-labeled N protein eluted from the lectin-Sepharose in the presence of GTP gamma S compared to membranes pretreated with either antagonist or agonist plus GTP. Our results therefore provide evidence that the same guanine nucleotide-binding protein that associates with the beta-adrenergic receptor in the presence of agonist mediates adenylate cyclase activation.

Conditions have been developed for desensitizing the beta-adrenergic receptor-coupled adenylate cyclase of turkey erythrocytes in a cell-free system. Desensitization is observed when cell lysates are incubated with isoproterenol or cAMP analogs for 30 min at 37 degrees C. Maximally effective concentrations of isoproterenol produce a 41.0 +/- 1.55% loss of iosproterenol-stimulated and a 15.0 +/- 2.35% loss of fluoride-stimulated enzyme activity. cAMP causes a 26.5 +/- 1.5% fall in isoproterenol-stimulated and a 21.5 +/- 4.4% fall in fluoride-sensitive activity. Desensitization by isoproterenol is dose-dependent, stereospecific, and blocked by the beta-adrenergic antagonist propranolol. Cell-free desensitization required ATP, Mg2+, and factor(s) present in the soluble fraction of the cell. Nonphosphorylating analogs of ATP did not support desensitization. Desensitization by agonist or cAMP in the cell-free system caused structural alterations in the beta-adrenergic receptor peptides apparent as an altered mobility of the photoaffinity labeled receptor peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As with the desensitization reaction, supernatant factors and ATP were also required for the agonist or cAMP-promoted receptor alterations. These data indicate that beta-adrenergic agonists promote a cAMP-mediated process which leads to receptor alterations and desensitization. The reactions involved in this process require ATP and soluble cellular factors. Additional processes must also occur to account for decreases in fluoride-sensitive enzyme activity. The availability of this cell-free system should facilitate elucidation of the molecular mechanisms involved in these processes.

In mouse 3T3-L1 preadipocytes, the glucocorticoid dexamethasone has been shown to promote a switch in beta-adrenoceptor subtype expression from beta 1 to beta 2 and to increase the total number of beta-adrenoceptors. The present study demonstrates that sodium butyrate also modulates beta-adrenoceptor expression in these cells. Incubation of preadipocytes with 2-10 mM butyrate for 24-48 h promoted a dose- and time-dependent switch in beta-adrenoceptor subtype from a near equal mixture of beta 1 and beta 2 to greater than 85% beta 2 and caused an approximate doubling of the receptor number. beta-Adrenoceptors were assayed in membranes prepared from 3T3-L1 cells using the radiolabeled antagonist [125I]iodocyanopindolol and the beta 2-selective antagonist ICI 118.551. Other short chain acids were not as effective as butyrate in promoting changes in beta-adrenoceptor expression. Cycloheximide (1.0 microgram/ml) inhibited the effects of butyrate on both beta-adrenoceptor subtype and number. Alterations in beta-adrenoceptor phenotype promoted by either butyrate or dexamethasone were functionally correlated with cAMP accumulation in these cells. Comparison of the effects of butyrate and dexamethasone on beta-adrenoceptor expression suggests that these two agents regulate beta-adrenoceptors by different mechanisms.

The tissue distribution of pertussis toxin-sensitive GTP-binding proteins was examined using specific antibodies raised against the purified alpha-subunit of G0 from bovine brain or against synthetic peptides predicted from cDNAs for distinct Gi subtypes. GTP-binding proteins were partially purified from membrane fractions prepared from rabbit tissues including brain, heart, liver, lung, erythrocytes and neutrophils. Brain contained both G0 and Gi1. Gi1 was also found to be abundant in heart. All peripheral tissues contained readily detectable amounts of Gi2, whereas only barely detectable amounts of Gi2 were found in brain. Gi3 was found to be prominent in erythrocytes and exists as a minor component of G proteins in neutrophils and liver. Thus, Gi2 appears to be widely disseminated in peripheral rabbit tissues, while other pertussis toxin substrates are more limited in their distribution.

A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [3H]-leukotriene D4 [( 3H]-LTD4), in the presence or absence of unlabeled LTD4, the diastereoisomer of LTD4 (5R,6S-LTD4), leukotriene E4 (LTE4) and the end-organ antagonist, FPL 55712, we have identified specific binding sites for [3H]-LTD4 in the crude membrane fraction isolated from guinea pig lung. The time required for [3H]-LTD4 binding to reach equilibrium was approximately 20 to 25 min at 37 degrees C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [3H]-LTD4 to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (Bmax), derived from Scatchard analysis, was approximately 320 +/- 200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5 +/- 4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 competed with [3H]-LTD4. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with [3H]-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.

Isoproterenol-induced desensitization of turkey erythrocyte adenylate cyclase is accompanied (1) by a decrease in the mobility of beta-adrenergic receptor proteins, specifically photoaffinity labeled with 125I-(p-azidobenzyl)carazolol (125I-PABC), on sodium dodecyl sulfate (SDS)-polyacrylamide gels and (2) by a 2-3-fold increase in phosphate incorporation into the beta receptor [Stadel, J.M., Nambi, P., Shorr, R. G. L., Sawyer, D. F., Caron, M. G., & Lefkowitz, R. J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3173]. Analysis of 32P-labeled beta receptors partially purified by affinity chromatography and subsequently hydrolyzed in 6 N HCl revealed that the beta receptor from control erythrocytes contained only phosphoserine and that agonist-promoted phosphorylation of the receptor in desensitized cells occurred on serine residues. Comparison of limited-digest peptide maps of 125I-PABC-labeled beta receptors from control and desensitized erythrocytes reveals distinctly different sensitivities of the two beta receptors to cleavage by chymotrypsin and Staphylococcus aureus protease. The altered mobility of the 125I-PABC-labeled beta receptor from desensitized erythrocytes was eliminated when 5 M urea was included in the SDS-polyacrylamide gels. Limited-digest peptide mapping of 32P-labeled beta receptors from control and desensitized cells with the protease papain identified a unique phosphorylated peptide in desensitized preparations. Our results are consistent with the hypothesis that the altered mobility of beta-receptor proteins on SDS gels following desensitization is due to changes in conformation promoted by prolonged exposure to agonists.

The predominant guanine nucleotide-binding protein (G-protein) of bovine lung membranes, termed GL, has been purified and compared biochemically, immunochemically and functionally with Gi and Go purified from rabbit brain. The purified GL appeared to have a similar subunit structure to Gi and Go, being composed of alpha, beta and possibly gamma subunits. On Coomassie Blue-stained SDS/polyacrylamide gels and immunoblots, the alpha subunit of GL (GL alpha) displayed an intermediate mobility (40 kDa) between those of Gi and Go (Gi alpha and Go alpha). GL alpha was [32P]ADP-ribosylated in the presence of pertussis toxin and [32P]NAD+. Analysis of [32P]ADP-ribosylated alpha subunits by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing showed that GL alpha was distinct from Gi alpha and Go alpha, but very similar to the predominant G-protein in neutrophil membranes. Immunochemical characterization also revealed that GL was distinct from Gi and Go, but was indistinguishable from the G-protein of neutrophils, which has been tentatively identified as Gi2 [Goldsmith, Gierschik, Milligan, Unson, Vinitsky, Maleck & Spiegel (1987) J. Biol. Chem. 262, 14683-14688]. In functional studies, higher Mg2+ concentrations were required for guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to GL than were required for nucleotide binding to Go, whereas Gi showed a Mg2+-dependence similar to that of GL. The kinetics of GTP[35S] binding to GL was quite different from those of Gi and Go; t1/2 values of maximal binding were 30, 15 and 5 min respectively. In contrast, the rate of hydrolysis of [gamma-32P]GTP by GL (t1/2 approximately 1 min) was approx. 4 times faster than that by Gi or Go. These results indicated that the predominant G-protein purified from lung is structurally and functionally distinct from Gi and Go of brain, but structurally indistinguishable from Gi2 of neutrophils.