A new phospholipase-C-calcium signalling pathway mediated by cyclic AMP and a Rap GTPase.

Stimulation of phosphoinositide-hydrolysing phospholipase C (PLC) generating inositol-1,4,5-trisphosphate is a major calcium signalling pathway used by a wide variety of membrane receptors, activating distinct PLC-beta or PLC-gamma isoforms. Here we report a new PLC and calcium signalling pathway that is triggered by cyclic AMP (cAMP) and mediated by a small GTPase of the Rap family. Activation of the adenylyl cyclase-coupled beta2-adrenoceptor expressed in HEK-293 cells or the endogenous receptor for prostaglandin E1 in N1E-115 neuroblastoma cells induced calcium mobilization and PLC stimulation, seemingly caused by cAMP formation, but was independent of protein kinase A (PKA). We provide evidence that these receptor responses are mediated by a Rap GTPase, specifically Rap2B, activated by a guanine-nucleotide-exchange factor (Epac) regulated by cAMP, and involve the recently identified PLC-epsilon isoform.

A novel bifunctional phospholipase c that is regulated by Galpha 12 and stimulates the Ras/mitogen-activated protein kinase pathway.

Three families of phospholipase C (PI-PLCbeta, gamma, and delta) are known to catalyze the hydrolysis of polyphosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PIP(2)) to generate the second messengers inositol 1,4,5 trisphosphate and diacylglycerol, leading to a cascade of intracellular responses that result in cell growth, cell differentiation, and gene expression. Here we describe the founding member of a novel, structurally distinct fourth family of PI-PLC. PLCepsilon not only contains conserved catalytic (X and Y) and regulatory domains (C2) common to other eukaryotic PLCs, but also contains two Ras-associating (RA) domains and a Ras guanine nucleotide exchange factor (RasGEF) motif. PLCepsilon hydrolyzes PIP(2), and this activity is stimulated selectively by a constitutively active form of the heterotrimeric G protein Galpha(12). PLCepsilon and a mutant (H1144L) incapable of hydrolyzing phosphoinositides promote formation of GTP-Ras. Thus PLCepsilon is a RasGEF. PLCepsilon, the mutant H1144L, and the isolated GEF domain activate the mitogen-activated protein kinase pathway in a manner dependent on Ras but independent of PIP(2) hydrolysis. Our findings demonstrate that PLCepsilon is a novel bifunctional enzyme that is regulated by the heterotrimeric G protein Galpha(12) and activates the small G protein Ras/mitogen-activated protein kinase signaling pathway.

Functional comparison of transmyocardial revascularization by mechanical and laser means.

BACKGROUND: As a result of the clinical benefit observed in angina patients treated by transmyocardial revascularization (TMR) with a laser, interest in mechanical TMR has been renewed. Although the injury induced by mechanical TMR is similar to laser TMR, the resultant impact on myocardial contractility is unknown. The purpose of this study was to determine whether mechanical TMR improves ventricular function as compared with laser TMR in chronically ischemic myocardium. METHODS: After establishing an area of chronic myocardial ischemia, 25 domestic pigs were randomized to treatment by: excimer laser (group I), a hot needle (50 degrees C) (group II), a normothermic needle (group III), an ultrasonic needle (40 KHz) (group IV), or no treatment (group V). All devices create a transmural channel of the same diameter; 22 +/- 1 transmural channels were created in each animal. Regional myocardial contractility was assessed by measuring ventricular wall thickening at rest and with dobutamine stress echocardiography. Six weeks after revascularization, the animals were restudied at rest and with stress. Postsacrifice and histologic analysis of angiogenesis and TMR effects was then assessed. RESULTS: Laser TMR provided significant recovery of ischemic myocardial function. This improvement in contractility after laser TMR was a 75% increase over the baseline function of the ischemic zone (p < 0.01). Mechanical TMR provided no significant improvement in function posttreatment. In fact, TMR achieved with an ultrasonic needle demonstrated a 40% worsening of the contractility versus the pretreatment baseline (p < 0.05). Histologic analysis demonstrated a significant increase in new blood vessels in the ischemic zone after laser TMR, which was not demonstrated for any of the other groups (p < 0.05). Additionally, evaluation of the mechanical TMR channels demonstrated significant scarring, which correlated with the functional results. CONCLUSIONS: Using devices to create an injury analogous to the laser, mechanical TMR failed to improve the function of chronically ischemic myocardium. Only laser TMR significantly improved myocardial function.

Contrast-enhanced magnetic resonance imaging of myocardium at risk: distinction between reversible and irreversible injury throughout infarct healing.

OBJECTIVES: We sought to determine the relationship of delayed hyperenhancement by contrast magnetic resonance imaging (MRI) to viable and nonviable myocardium within the region at risk throughout infarct healing. BACKGROUND: The relationship of delayed MRI contrast enhancement patterns to injured but viable myocardium within the ischemic bed at risk has not been established. METHODS: We compared in vivo and ex vivo MRI contrast enhancement to histopathologic tissue sections encompassing the entire left ventricle in dogs (n = 24) subjected to infarction with (n = 12) and without (n = 12) reperfusion at 4 h, 1 day, 3 days, 10 days, 4 weeks and 8 weeks. In vivo MR imaging was performed 30 min after contrast injection. RESULTS: The sizes and shapes of in vivo myocardial regions of elevated image intensity (828+/-132% of remote) were the same as those observed ex vivo (241 slices, r = 0.99, bias = 0.05+/-1.6% of left ventricle [LV]). Comparison of ex vivo MRI to triphenyltetrazolim chloride-stained sections demonstrated that the spatial extent of hyperenhancement was the same as the spatial extent ofinfarction at every stage of healing (510 slices, lowest r = 0.95, largest bias = 1.7+/-2.9% of LV). Conversely, hyperenhanced regions were smaller than the ischemic bed at risk defined by fluorescent microparticles at every stage of healing (239 slices, 35+/-24% of risk region, p<0.001). Image intensities of viable myocardium within the risk region were the same as those of remote, normal myocardium (102+/-9% of remote, p = NS). CONCLUSIONS: Delayed contrast enhancement by MRI distinguishes between viable and nonviable regions within the myocardium at risk throughout infarct healing.

Up-regulation of vascular endothelial growth factor mRNA and angiogenesis after transmyocardial laser revascularization.

BACKGROUND: Angiogenesis has been proposed as a potential mechanism whereby transmyocardial laser revascularization (TMLR) has provided clinical relief of angina. Experimental work has found histologic evidence supporting this, as well as an improved response when angiogenic growth factors have been added to TMLR. The purpose of this study was to demonstrate that the molecular response to TMLR was an increase in the production of endogenous vascular endothelial growth factor to promote angiogenesis. METHODS: Ameroid constrictors were placed on the proximal circumflex artery in 12 domestic pigs. After a chronic ischemic zone was established the animals were randomly divided into two groups. In the TMLR group the ischemic zone was treated with carbon dioxide laser. In the control group the ischemic zone was untreated. Six weeks later the animals were sacrificed, and sections from the ischemic zone and the nonischemic zone were submitted for immunohistochemical, histologic, and molecular analysis. Messenger RNA was obtained from northern blot analysis after being probed with vascular endothelial growth factor. RESULTS: There was a twofold increase in the vascular endothelial growth factor messenger RNA in the ischemic zone of the TMLR group compared with the control group. Additionally, there was a threefold increase in the number of new blood vessels in the ischemic zone of the TMLR group compared with the control group. CONCLUSIONS: Transmyocardial laser revascularization promotes angiogenesis by upregulation of vascular endothelial growth factor. The resulting angiogenesis could be the principle mechanism for the clinical efficacy of TMLR.

Interactions of G(h)/transglutaminase with phospholipase Cdelta1 and with GTP.

The inositol phosphate hydrolyzing activity of human phospholipase Cdelta1 (PLCdelta1) is markedly inhibited when the enzyme is coexpressed with the human heart G(h)/transglutaminase (TG) in human embryonic kidney cells. Because the cotransfection does not affect the amount of PLCdelta1 in the cells, the depression of phospholipase activity probably is a result of a direct interaction between the two proteins. An ELISA procedure was employed to document the associations of purified TG preparations from a variety of tissues (human red cells, rabbit lens, guinea pig liver) with PLCdelta1. Nucleotides (GTP > GDP > ATP > GMP = ADP, in order of decreasing efficiency) interfered with the formation of the PLCdelta1:TG complex. A conformational change in the TG partner, occurring with nucleotide binding, is thought to be responsible for dissociating the two proteins. The structural rearrangement produces a remarkable shift in the anodic mobility of TG in electrophoresis: TG(slow) + GTP -->/<-- [TG:GTP](fast). Altogether, our findings indicate that GTP controls PLCdelta1 activity by releasing this protein from an inhibitory association with G(h)/transglutaminase.

Activation of phospholipase C delta1 through C2 domain by a Ca(2+)-enzyme-phosphatidylserine ternary complex.

The concentration of free Ca(2+) and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C delta1 (PLCdelta1). The rate of PLCdelta1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated 20-fold by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca(2+) concentration required for half-maximal activation of PLCdelta1 from 5.4 to 0.5 microM. In the presence of Ca(2+), PLCdelta1 specifically bound to PS/PC but not to PA/PC vesicles in a dose-dependent and saturable manner. Ca(2+) also bound to PLCdelta1 and required the presence of PS/PC vesicles but not PA/PC vesicles. The free Ca(2+) concentration required for half-maximal Ca(2+) binding was estimated to be 8 microM. Surface dilution kinetic analysis revealed that the K(m) was reduced 20-fold by the presence of 25 mol % PS, whereas V(max) and K(d) were unaffected. Deletion of amino acid residues 646-654 from the C2 domain of PLCdelta1 impaired Ca(2+) binding and reduced its stimulation and binding by PS. Taken together, the results suggest that the formation of an enzyme-Ca(2+)-PS ternary complex through the C2 domain increases the affinity for substrate and consequently leads to enzyme activation.

A single amino acid substitution in the pleckstrin homology domain of phospholipase C delta1 enhances the rate of substrate hydrolysis.

The pleckstrin homology (PH) domain has been postulated to serve as an anchor for enzymes that operate at a lipid/water interface. To understand further the relationship between the PH domain and enzyme activity, a phospholipase C (PLC) delta1/PH domain enhancement-of-activity mutant was generated. A lysine residue was substituted for glutamic acid in the PH domain of PLC delta1 at position 54 (E54K). Purified native and mutant enzymes were characterized using a phosphatidylinositol 4,5-bisphosphate (PI(4, 5)P2)/dodecyl maltoside mixed micelle assay and kinetics measured according to the dual phospholipid model of Dennis and co-workers (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739; Carmen, G. M., Deems, R. A., and Dennis, E. A. (1995) J. Biol. Chem. 270, 18711-18714). Our results show that both PLC delta1 and E54K bind phosphatidylinositol bisphosphate cooperatively (Hill coefficients, n = 2.2 +/- 0.2 and 2.0 +/- 0.1, respectively). However, E54K shows a dramatically increased rate of (PI(4, 5)P2)-stimulated PI(4,5)P2 hydrolysis (interfacial Vmax for PLC delta1 = 4.9 +/- 0.3 micromol/min/mg and for E54K = 31 +/- 3 micromol/min/mg) as well as PI hydrolysis (Vmax for PLC delta1 = 27 +/- 3.4 nmol/min/mg and for E54K = 95 +/- 12 nmol/min/mg). In the absence of PI(4,5)P2 both native and mutant enzyme hydrolyze PI at similar rates. E54K also has a higher affinity for micellar substrate (equilibrium dissociation constant, Ks = 85 +/- 36 microM for E54K and 210 +/- 48 microM for PLC delta1). Centrifugation binding assays using large unilamelar phospholipid vesicles confirm that E54K binds PI(4,5)P2 with higher affinity than native enzyme. E54K is more active even though the interfacial Michaelis constant (Km) for E54K (0.034 +/- 0.01 mol fraction PI(4,5)P2) is higher than the Km for native enzyme (0.012 +/- 0.002 mol fraction PI(4,5)P2). D-Inositol trisphosphate is less potent at inhibiting E54K PI(4,5)P2 hydrolysis compared with native enzyme. These results demonstrate that a single amino acid substitution in the PH domain of PLC delta1 can dramatically enhance enzyme activity. Additionally, the marked increase in Vmax for E54K argues for a direct role of PH domains in regulating catalysis by allosteric modulation of enzyme structure.

Phosphatidylinositol 4,5-bisphosphate binding to the pleckstrin homology domain of phospholipase C-delta1 enhances enzyme activity.

The pleckstrin homology (PH) domain is a newly recognized protein module believed to play an important role in signal transduction. While the tertiary structures of several PH domains have been determined, some co-complexed with ligands, the function of this domain remains elusive. In this report, the PH domain located in the N terminus of human phospholipase C-delta1 (PLCdelta1) was found to regulate enzyme activity. The hydrolysis of phosphatidylinositol (PI) was stimulated by phosphatidylinositol 4,5-bisphosphate (PIP2) in a dose-dependent manner with an EC50 = 1 microM (0.3 mol%), up to 9-fold higher when 5 microM (1.5 mol%) of PIP2 was incorporated into the PI/phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (30 microM of PI with a molar ratio of PI:PS:PC = 1:5:5). Stimulation was specific for PIP2, since other anionic phospholipids including phosphatidylinositol 4-phosphate had no stimulatory effect. PIP2-mediated stimulation was, however, inhibited by inositol 1,4, 5-triphosphate (IP3) in a dose-dependent manner, suggesting a modulatory role for this inositol. When a nested set of PH domain deletions up to 70 amino acids from the N terminus of PLCdelta1 were constructed, the deletion mutant enzymes all catalyzed the hydrolysis of the micelle forms of PI and PIP2 with specific activities comparable with those of the wild type enzyme. However, the stimulatory effect of PIP2 was greatly diminished when more than 20 amino acid residues were deleted from the N terminus. To identify the specific residues involved in PIP2-mediated enzyme activation, amino acids with functional side chains between residues 20 and 40 were individually changed to glycine. While all these mutations had little effect on the ability of the enzyme to catalyze the hydrolysis of PI or PIP2 micelles, the catalytic activity of mutants K24G, K30G, K32G, R38G, or W36G was markedly unresponsive to PIP2. Analysis of PIP2-stimulated PI hydrolysis by a dual substrate binding model of catalysis revealed that the micellar dissociation constant (Ks) of PLCdelta1 for the PI/PS/PC vesicles was reduced from 558 microM to 53 microM, and the interfacial Michaelis constant (Km) was reduced from 0.21 to 0.06 by PIP2. The maximum rate of PI hydrolysis (Vmax) was not affected by PIP2. These results demonstrate that a major function of the PH domain of PLCdelta1 is to modulate enzyme activity. Further, our results identify PIP2 as a functional ligand for a PH domain and suggest a general mechanism for the regulation of other proteins by PIP2.

Cloning and identification of amino acid residues of human phospholipase C delta 1 essential for catalysis.

In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding. These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC.

Lipid-mediated regulation of G protein-coupled receptor kinases 2 and 3.

G protein-coupled receptor-mediated signaling is attenuated by a process referred to as desensitization, wherein agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs) is proposed to be a key initial event. However, mechanisms that activate GRKs are not fully understood. In one scenario, beta gamma-subunits of G proteins (G beta gamma) activate certain GRKs (beta-adrenergic receptor kinases 1 and 2, or GRK2 and GRK3), via a pleckstrin homology domain in the COOH terminus. This interaction has been proposed to translocate cytosolic beta-adrenergic receptor kinases (beta ARKs) to the plasma membrane and facilitate interaction with receptor substrates. Here, we report a novel finding that membrane lipids modulate beta ARK activity in vitro in a manner that is analogous and competitive with G beta gamma. Several lipids, including phosphatidylserine (PS), stimulated, whereas phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine receptors. Furthermore, both PS and phosphatidylinositol 4,5-bisphosphate specifically bound to beta ARK1, whereas phosphatidylcholine, a lipid that did not modulate beta ARK activity, did not bind to beta ARK1. The lipid regulation of beta ARKs did not occur via a modulation of its autophosphorylation state. PS- and G beta gamma-mediated stimulation of beta ARK1 was compared and found strikingly similar; moreover, their effects together were not additive (except at initial stages of reaction), which suggests that PS and G beta gamma employed a common interaction and activation mechanism with the kinase. The effects of these lipids were prevented by two well known G beta gamma-binding proteins, phosducin and GST-beta ARK-(466-689) fusion protein, suggesting that the G beta gamma-binding domain (possibly the pleckstrin homology domain) of the GRKs is also a site for lipid:protein interaction. We submit the intriguing possibility that both lipids and G proteins co-regulate the function of GRKs.

Species orthologs of the alpha-2A adrenergic receptor: the pharmacological properties of the bovine and rat receptors differ from the human and porcine receptors.

Four pharmacological subtypes of the alpha-2 adrenergic receptor have been identified; however, only three subtypes exist in any given species. Although the alpha-2A adrenergic receptor, as defined by the human platelet, and the alpha-2D receptor, as defined in the bovine pineal, have very different pharmacological characteristics, they are more similar to each other than either is to the alpha-2B or alpha-2C subtype. The human alpha-2-C10 clone (alpha-2A) and the rat RG20 clone have an 89% identity in their predicted amino acid sequence and are considered to be species orthologs. Although the expressed RG20 clone appears to have alpha-2D pharmacology, a careful comparison of its pharmacological characteristics with the bovine pineal has not been reported previously. Based on the pKi values of a panel of 13 alpha-2 adrenergic agents that have been used previously to compare the alpha-2A, alpha-2B and alpha-2C subtypes, the pharmacological characteristics of the bovine pineal alpha-2D receptor appear to be very similar to the rat RG20 clone (correlation coefficient, r, of 0.93). The porcine ortholog of the human alpha-2-C10 receptor has pharmacological characteristics identical to the human alpha-2A receptor (r = 0.99). Because of its higher affinity for the alpha-2D receptor, [3H]RX821002 is a better radioligand than [3H]rauwolscine for studying this receptor subtype.

Distribution of alpha 2-adrenergic receptor subtype gene expression in rat brain.

alpha 2-Adrenergic receptors in brain are important presynaptic modulators of central noradrenergic function (autoreceptors) and postsynaptic mediators of many of the widespread effects of catecholamines and related drugs. alpha 2-Adrenergic agonists are currently used as antihypertensives and preanesthetic agents, but new subtype-selective alpha 2-adrenoceptor agonists and antagonists have additional therapeutic application potential. Three genes encoding specific alpha 2-adrenoceptor subtypes (alpha 2A, alpha 2B, and alpha 2C) have been isolated and characterized. RNA blotting indicates that all three are expressed in rat brain. This study used in situ hybridization with 35S-labeled RNA probes to map the distribution of alpha 2-adrenoceptor subtype gene expression in rat brain. alpha 2A mRNA was most abundant in the locus coeruleus, but was also widely distributed in the brain stem, cerebral cortex, septum, hypothalamus, hippocampus and amygdala. alpha 2B mRNA was observed only in the thalamus. alpha 2C mRNA was mainly localized to the basal ganglia, olfactory tubercle, hippocampus, and cerebral cortex. These mRNA distributions largely agree with previous findings on the alpha 2-adrenoceptor distributions in the rat brain, but suggest that the localization patterns for each receptor subtype are unique. The expression of alpha 2A mRNA in noradrenergic neurons indicates that this subtype mediates presynaptic autoreceptor functions. Furthermore, the localization of alpha 2A mRNA in noradrenergic projection areas suggests that this receptor may also have an important role in mediating postsynaptic effects. The precise physiological and pharmacological roles of the alpha 2-adrenoceptor subtypes are still largely unknown, but it is expected that in situ hybridization coupled to various methods to identify the transmitter phenotypes of the subtype-expressing neurons will help to clarify these important issues in the near future.

Identification, quantification, and localization of mRNA for three distinct alpha 1 adrenergic receptor subtypes in human prostate.

The dynamic component of bladder outlet obstruction caused by benign prostatic hyperplasia (BPH) is regulated by alpha 1 adrenergic receptors (alpha 1-AR) located in the prostatic stroma. Recently two alpha 1-AR subtypes (alpha 1A, alpha 1B) have been identified in the human prostate by both functional and pharmacological assays. However, the presence of the alpha 1C subtype has not been evaluated, presumably due to the lack of availability of selective ligands for this receptor subtype. We have used molecular techniques to investigate the mRNA expression of all three alpha 1-AR subtypes in the human prostate. RNA extracted from the prostate gland of 15 patients was used in ribonuclease protection assays to identify the expression of three alpha 1-AR subtype mRNAs. Quantitative solution hybridization assays further identify the predominant subtype of alpha 1-AR mRNA to be the alpha 1C, which represents approximately 70% of the total alpha 1-AR mRNA in the human prostate. Furthermore, in situ hybridization localizes the alpha 1C AR mRNA predominantly to the stromal compartment. The identification of a predominant alpha 1-AR mRNA in human prostate identifies a potential need for subtype selective pharmaceutical agents. These agents could be very important clinically in the treatment of diseases such as BPH.

Pharmacologic characterization of cloned alpha 1-adrenoceptor subtypes: selective antagonists suggest the existence of a fourth subtype.

In membranes prepared from Cos-7 or HeLa cells expressing one of three individual cloned alpha 1-adrenoceptor subtypes, competition with 2-[(beta-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl]-tetralone ([125I]HEAT) by the selective compounds [+]-niguldipine, 5-methyl-urapidil, and benoxathian reveals high affinity for the cloned alpha 1C-adrenoceptor subtype and low affinity for both the cloned alpha 1A-adrenoceptor and alpha 1B-adrenoceptor. Competition with [125I]HEAT by spiperone revealed high affinity for the cloned alpha 1C-adrenoceptor, intermediate affinity for the cloned alpha 1B-adrenoceptor, and low affinity for the cloned alpha 1A-adrenoceptor. Combining pharmacological properties previously described for alpha 1-adrenoceptor subtypes in rat membranes and here described from cloned receptors, these data suggest the existence of a fourth distinct alpha 1-adrenoceptor subtype.

Pharmacological characteristics of alpha 2-adrenergic receptors: comparison of pharmacologically defined subtypes with subtypes identified by molecular cloning.

On the basis of extensive radioligand data and more limited functional data, three pharmacological subtypes of alpha 2-adrenergic receptors have been identified. More recently, three human genes or cDNAs for alpha 2-adrenergic receptors have been identified by molecular cloning. The relationship, however, among the pharmacologically defined subtypes and those identified by molecular cloning has not been clear. In order to resolve this issue, we have compared the pharmacological characteristics of the receptors identified by molecular cloning and expressed in COS-7 cells with the characteristics of the pharmacologically defined receptors in their respective prototypic tissue or cell line. The affinities (Ki values) of 12 subtype-selective alpha 2-adrenergic antagonists were determined for the alpha 2 receptor in the six preparations, by radioligand binding. Correlation analyses of the pKi values indicate that the alpha 2A subtype, as defined in the HT29 cell line, the alpha 2B receptor of the neonatal rat lung, and the alpha 2C subtype, as defined in an oppossum kidney cell line, correspond to the cloned human alpha 2-C10, alpha 2-C2, and alpha 2-C4 receptor subtypes, respectively.

Molecular cloning and expression of the cDNA for the alpha 1A-adrenergic receptor. The gene for which is located on human chromosome 5.

Pharmacological and molecular cloning studies have demonstrated heterogeneity of alpha 1-adrenergic receptors. We have now cloned two alpha 1-adrenergic receptors from a rat cerebral cortex cDNA library, using the hamster alpha 1B-adrenergic receptor as a probe. The deduced amino acid sequence of clone RA42 encodes a protein of 560 amino acids whose putative topology is similar to that of the family of G-protein-coupled receptors. The primary structure though most closely resembles that of an alpha 1-adrenergic receptor, having approximately 73% amino acid identity in the putative transmembrane domains with the previously isolated hamster alpha 1B receptor. Analysis of the ligand binding properties of RA42 expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 1-adrenergic receptor pharmacology. High affinity for the antagonist WB4101 and agonists phenylephrine and methoxamine suggests that cDNA RA42 encodes the alpha 1A receptor subtype. Northern blot analysis of various rat tissues also shows the distribution expected of the alpha 1A receptor subtype with abundant expression in vas deferens followed by hippocampus, cerebral cortex, aorta, brainstem, heart and spleen. The second alpha 1-adrenergic receptor cloned represents the rat homolog of the hamster alpha 1B subtype. Expression of mRNA for this receptor is strongly detected in liver followed by heart, cerebral cortex, brain stem, kidney, lung, and spleen. This study provides definitive evidence for the existence of three alpha 1-adrenergic receptor subtypes.