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.     

Sequence and Regional Distribution of the mRNA Encoding the α2 Polypeptide of Rat γ-Aminobutyric AcidA Receptors

A cDNA from a rat hippocampal cDNA library encodes an isoform of the alpha polypeptide of the gamma-aminobutyric acid (GABA)/benzodiazepine (BZ) receptor. Its deduced amino acid sequence is 96% identical to that of the alpha 2 polypeptide of the bovine GABAA receptor. The polypeptide has features shared by all previously reported GABAA receptor alpha polypeptides and shares 71-76% identity with previously described rat alpha polypeptides. Most of the differences lie in the presumed extracellular and intracellular domains. On Northern blots, the alpha 2 cDNA detects two mRNAs, which are found in cortex, hippocampus, and striatum, brain regions enriched in pharmacologically defined "BZ type II" receptors. Other workers have previously shown that the alpha polypeptides of the GABAA receptor largely determine the BZ binding properties of reconstituted receptors. The distribution of alpha 2 mRNAs in rat brain suggests that the alpha 2 subunit may indeed be involved in the BZ type II receptors.     

Molecular cloning and pharmacological characterization of the canine B1 and B2 bradykinin receptors

The dog is a valuable animal model in the study of the physiological role of both the B1 and B2 bradykinin receptors. To more thoroughly characterize the pharmacological properties of the canine kinin receptors we isolated the cDNA sequence encoding the B1 and B2 bradykinin receptor subtypes and overexpressed them in Chinese hamster ovary (CHO) cells. The cDNA sequence of the canine B1 bradykinin receptor encodes a protein comprised of 350 amino acids that is 76% identical to the human B1 bradykinin receptor. The cDNA sequence of the canine B2 bradykinin receptor encodes a protein of 392 amino acids that is 81% identical to the human B2 bradykinin receptor. The amino acid sequence of the canine B1 and B2 receptors are 35% identical. Pharmacological studies of the cloned receptors revealed that the agonist affinity of the dog B1 receptor is similar to the rodent B1 receptors, and differs from the human form in that there is no preference for the presence of the N-terminal Lys residue of [des-Arg10]Lys-bradykinin. Significantly, the B1 receptor antagonist [des-Arg9,Leu8]BK behaves as partial agonist on the cloned dog B1 receptor. The dog B2 receptor exhibits the 'classical' pharmacological properties of this receptor subtype.     

Molecular cloning and expression of the cDNA for a novel A2-adenosine receptor subtype.

A novel adenosine receptor subtype has been cloned from a rat brain cDNA library using a probe generated by the polymerase chain reaction. The cDNA, designated RFL9, encodes a protein of 332 amino acids. The structure of RFL9 is most similar to that of the recently cloned rat A2-adenosine receptor, with a sequence identity of 73% within the presumed seven transmembrane domains. Expression of RFL9 in COS-6M cells resulted in ligand binding and functional activity characteristics of an adenosine receptor that is coupled positively to adenylyl cyclase. Examination of the tissue distribution of RFL9 mRNA by Northern blot analysis showed a restricted distribution with highest levels expressed in large intestine, cecum, and urinary bladder; this pattern was distinct from that of either the A1- or A2-adenosine receptor mRNAs. In situ hybridization studies of RFL9 mRNA showed no specific hybridization pattern in brain, but a hybridization signal was readily observed in the hypophyseal pars tuberalis. Thus, RFL9 encodes a novel A2-adenosine receptor subtype.     

Galphaq-dependent activation of mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase cascade.

G-protein-coupled receptors (GPCRs) typically activate c-Jun N-terminal kinase (JNK) through the G protein betagamma subunit (Gbetagamma), in a manner dependent on Rho family small GTPases, in mammalian cells. Here we show that JNK activation by the prototypic Gq-coupled alpha1B-adrenergic receptor is mediated by the alpha subunit of Gq (Galphaq), not by Gbetagamma, using a transient transfection system in human embryonic kidney cells. JNK activation by the alpha1B-adrenergic receptor/Galphaq was selectively mediated by mitogen-activated protein kinase kinase 4 (MKK4), but not MKK7. Also, MKK4 activation by the alpha1B-adrenergic receptor/Galphaq required c-Src and Rho family small GTPases. Furthermore, activation of the alpha1B-adrenergic receptor stimulated JNK activity through Src family tyrosine kinases and Rho family small GTPases in hamster smooth muscle cells that natively express the alpha1B-adrenergic receptor. Together, these results suggest that the alpha1B-adrenergic receptor/Galphaq may up-regulate JNK activity through a MKK4 pathway dependent on c-Src and Rho family small GTPases in mammalian cells.     

Cloning, sequencing, and expression of the gene encoding the porcine alpha 2-adrenergic receptor. Allosteric modulation by Na+, H+, and amiloride analogs

The gene for an alpha 2-adrenergic receptor has been cloned from a porcine genomic library, using as a probe a 0.95-kilobase Pst fragment of the gene for the human platelet alpha 2-adrenergic receptor. The identity of the cloned porcine gene was confirmed initially on the basis of partial amino acid sequence information obtained following cyanogen bromide digestion of homogeneous preparations of porcine brain alpha 2-adrenergic receptors. The deduced amino acid sequence for the porcine receptor, when compared to other members of the family of guanine nucleotide-binding protein-coupled receptors, shares the same overall structural characteristics and most closely resembles the human platelet C10 alpha 2-adrenergic receptor (greater than 93% homology). The putative porcine alpha 2-receptor gene was expressed in the COS-M6 cell line. Transfected cells display saturable [3H]yohimbine binding. The KD for [3H]yohimbine, determined in digitonin-solubilized preparations, is 5.8 nM. The selectivity of agonists and antagonists in competing for [3H]yohimbine binding to membranes prepared from the transfected cells is characteristic of the alpha 2A subtype of adrenergic receptors. The porcine alpha 2-receptor also was expressed permanently in LLC-PK1 porcine kidney cells at a level of 100 pmol/mg protein. The alpha 2-agonist UK14304 is able to attenuate forskolin or vasopressin-stimulated cAMP accumulation by at least 50% in these cells. Allosteric modulation of [3H] yohimbine binding by Na+, H+, and 5-amino-substituted analogs of amiloride also was demonstrated for the alpha 2-receptor expressed in COS-M6 cells. Moreover, these modulatory effects were quantitatively similar to those observed for homogeneous preparations of the alpha 2-receptor purified from porcine brain cortex. Retention of the effects of cations and amiloride analogs in transiently expressed alpha 2-receptors supports the interpretation that the allosteric sites for these agents reside in the alpha 2-receptor molecule itself.     

Molecular cloning and sequencing of a cDNA encoding a human alpha 1A adrenergic receptor.

DNA from a rat hippocampus cDNA library and sets of highly degenerate oligonucleotide primers directed toward conserved regions of previously cloned G-protein receptors were used in the polymerase chain reaction to selectively amplify and clone new members of this gene family. A human hippocampus cDNA library was screened with a 610 base pair fragment generated by PCR and a cDNA clone, H318/3, was isolated. The deduced amino acid sequence of this clone encoded a protein of 501 amino acids that showed strong sequence homology to previously cloned G-protein receptors. Nucleotide sequence analysis revealed clone H318/3 was 78% homologous to a rat alpha 1A adrenergic receptor with homology being 95% when comparisons were made in the region that lies between the first to the seventh transmembrane domains. Based on this high degree of sequence homology, we conclude that clone H318/3 represents a cDNA for a human alpha 1A adrenergic receptor.     

Localization of alpha1B-adrenergic receptor in female rat brain regions involved in stress and neuroendocrine function.

Activation of alpha1-adrenergic receptors has been linked to the control of blood pressure, neuroendocrine secretion, reproductive behavior and mood. The present study describes the distribution of alpha1B-adrenergic receptor immunoreactivity in female rat brain regions involved in stress and neuroendocrine function. The pattern of immunolabeling seen resembles that obtained in previous in situ hybridization studies. Several hypothalamic areas that control pituitary function showed intense fiber and/or cell immunolabeling, including the paraventricular nucleus of the hypothalamus, the supraoptic nucleus, and the median eminence. Some regions such as the arcuate nucleus, the median eminence, and dorsal hypothalamus exhibit intense labeling of axonal varicosities, while other regions exhibit only perikarya immunolabeling. alpha1B-adrenergic receptor immunoreactivity was also observed in large pyramidal neurons of layer V of the cerebral cortex, the frontal cortex showing a particularly strong immunoreactivity. Virtually all thalamic regions were labeled, especially the lateral and ventral areas. In addition, labeled cells were present in hippocampus, the medial septum, the horizontal and vertical limbs of the diagonal band of Broca, and the caudate putamen. Finally, some midbrain and hindbrain regions important for motor function were immunoreactive. Because ligands specific for alpha1-adrenergic receptor subtypes are not available, the present immunocytochemical study not only addresses the subcellular and regional distribution of alpha1B-adrenergic receptors but may also provide clues about receptor subtype-specific function.     

Cloning of the cDNA and Genes for the Hamster and Human β2-Adrenergic Receptors

Abstract

The adenylate cyclase-stimulatory β₂-adrenergic receptor has been purified to apparent homogeneity from hamster lung. Partial amino acid sequence obtained from isolated CNBr peptides was used to clone the gene and cDNA for this receptor. The predicted amino acid sequence for the hamster β₂-adrenergic receptor revealed that the protein consists of a single polypeptide chain of 418 aa with consensus N-glycosylation and phosphorylation sites predicted by previous in vitro data. The most striking feature of the receptor protein however, is that it contains seven stretches of hydrophobic residues similar to the proposed seven transmembrane segments of the light receptor rhodopsin. Significant amino acid homology (30-35%) can be found between the hamster β₂-adrenergic receptor and rhodopsin within these putative membrane spanning regions. Using a hamster β₂-adrenergic receptor probe, the gene and cDNA for the human β₂-adrenergic receptor were isolated, revealing a high degree of homology (87%) between the two proteins from different species. Unlike the genes encoding the family of opsin pigments, of which rhodopsin is a member, the genes encoding both hamster and human β₂-adrenergic receptors are devoid of introns in their coding as well as 5′ and 3′ untranslated nucleotide sequences. The cloning of the genes and the elucidation of the aa sequences for these G-protein coupled receptors should help to determine the structure-function as well as the evolutionary relationship of these proteins.     

Expression of mRNA of beta 1- and beta 2-adrenergic receptors in Xenopus oocytes results from structurally distinct receptor mRNAs

Poly(A)+-selected RNA prepared from cells or tissues that express a homogeneous population of either beta 1- or beta 2-adrenergic receptors was isolated and then microinjected into Xenopus laevis oocytes. Following microinjection, the expression of beta-adrenergic receptors was assessed by equilibrium radioligand binding analysis using the antagonist ligand [3H]dihydroalprenolol. The pharmacology of the newly- expressed beta-adrenergic receptors in oocyte membranes was the same as that of the original tissue used as a source of RNA. Hybridization of nick-translated cDNA of hamster beta 2-adrenergic receptor to poly(A)+-selected RNA from tissues containing beta 2-adrenergic receptors was to a mRNA species of 2.2 kilobases. In contrast, hybridization of the cDNA probe to poly(A)+-selected RNA from tissues containing beta 1-adrenergic receptors was to a mRNA species of 2.0 kilobases. A single-stranded fragment of hamster beta 2-adrenergic receptor cDNA corresponding to nucleotides 730-886 was isolated and uniformly radiolabeled. This region of the gene is predicted to encode for the entire second exofacial loop (L4-5), the entire fifth transmembrane-spanning region, and the first 5 amino acid residues of the third cytoplasmic loop (L5-6) of the beta 2-adrenergic receptor. Hybridization at 48 and 56 degrees C of poly(A)+-selected RNA prepared from sources that express either beta 1 or beta 2-adrenergic receptors to the antisense orientation strand of this region of the beta 2-adrenergic receptor cDNA was followed by S1 endonuclease digestion of nonhybridized sequences. At 48 degrees C, S1-resistant hybrids from both sources of RNA protected the probe from S1 endonuclease digestion. At 56 degrees C, however, only the RNA prepared from the source of beta 2-adrenergic receptors protected the probe from S1 endonuclease digestion. These results demonstrate that the mRNAs encoding for the structurally homologous beta 1- and beta 2-adrenergic receptors are distinct in the pharmacological specificity of their translation products and in their size and structure.     

Imidazoline receptor antisera-selected (IRAS) cDNA: cloning and characterization

The imidazoline-1 receptor (IR1) is considered a novel target for drug discovery. Toward cloning an IR1, a truncated cDNA clone was isolated from a human hippocampal lambda gt11 cDNA expression library by relying on the selectivity of two antisera directed against candidate IR proteins. Amplification reactions were performed to extend the 5' and 3' ends of this cDNA, followed by end-to-end PCR and conventional cloning. The resultant 5131-basepair molecule, designated imidazoline receptor-antisera-selected (IRAS) cDNA, was shown to encode a 1504-amino acid protein (IRAS-1). No relation exists between the amino acid sequence of IRAS-1 and proteins known to bind imidazolines (e.g., it is not an alpha2-adrenoceptor or monoamine oxidase subtype). However, certain sequences within IRAS-1 are consistent with signaling motifs found in cytokine receptors, as previously suggested for an IR1. An acidic region in IRAS-1 having an amino acid sequence nearly identical to that of ryanodine receptors led to the demonstration that ruthenium red, a dye that binds the acidic region in ryanodine receptors, also stained IRAS-1 as a 167-kD band on SDS gels and inhibited radioligand binding of native I1 sites in untransfected PC-12 cells (a source of authentic I1 binding sites). Two epitope-selective antisera were also generated against IRAS-1, and both reacted with the same 167-kD band on Western blots. In a host-cell-specific manner, transfection of IRAS cDNA into Chinese hamster ovary cells led to high-affinity I1 binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS-1 is the first protein discovered with characteristics of an IR1.     

Hamster estrogen receptor cDNA: cloning and mRNA expression

Estrogen-induced hamster kidney tumor model serves as a useful model to study the biochemical and molecular mechanisms of hormonal carcinogenesis. In this model, we have demonstrated an increased expression of estrogen receptor mRNA and protein in estrogen-treated kidneys and in estrogen-induced tumors. The sequence information for hamster estrogen receptor gene is not known and has been investigated in this study. A hamster uterus cDNA library was constructed and the 5'-region of the hamster estrogen receptor cDNA cloned from this library using polymerase chain reaction (PCR) methodology. Additionally, hamster kidney polyadenylated RNA was reverse transcribed and PCR amplified using primers that were designed based on maximum homology between mouse, rat and human estrogen receptor cDNAs. These PCR amplified fragments were cloned into plasmid vectors and clones with the expected size of the insert subjected to Southern blot analysis using human estrogen receptor cDNA as a probe. The positive clones on Southern blot analysis and the PCR amplified products from these clones were subjected to DNA sequence analysis. Using this strategy, a full length, 1978 bp hamster estrogen receptor cDNA has been cloned which shows 87% homology with human, 90% with rat and 91% with mouse estrogen receptor cDNA. The deduced amino acid shares 88% homology with human, and 93% with rat and mouse estrogen receptors. Hamster estrogen receptor domain C (DNA binding domain) shows a 100% homology with a similar domain from mouse, rat, human, pig, sheep, horse and chicken estrogen receptor (Genebank reference ID: AF 181077).     

Molecular cloning and expression of an adenosine A2b receptor from human brain.

A novel receptor cDNA was isolated from a human hippocampal cDNA library. The encoded polypeptide contains structural features consistent with its classification as a G protein-coupled receptor and shares 45% homology with the human A1 and A2a adenosine receptors. Chinese hamster ovary K1 cells expressing this receptor showed marked stimulation of adenylate cyclase when treated with 1mM adenosine. There was no response to ligands selective for A1 and A2a receptors but the general adenosine agonist N-ethylcarboxyamidoadenosine (NECA) caused a 10 fold increase in cyclic AMP accumulation with an EC50 of approximately 0.9 microM. This effect was inhibited by the adenosine receptor antagonist theophylline. Specific binding of A1 and A2a selective agonists and NECA was not detected. It is proposed that the novel receptor is a human brain adenosine A2b receptor subtype.     

Pharmacological characterization of alpha1- and beta-adrenergic synergism of 5'DII activity in rat brown adipocytes

The role of adrenoceptor subtypes was studied in rat brown adipose tissue (BAT). The type II 5'-deiodinase (5'DII) was activated in response to simultaneous stimulation by beta3- and alpha1-adrenergic agonists, BRL 37344 or CGP 12177, and cirazoline, in brown adipocytes. Inhibition of the alpha1- and beta-adrenergic phenylephrine-stimulated 5'DII activity was obtained by the alpha1-adrenergic antagonists in the order of prazosin >/= wb 4101 > 5-methylurapidil. In comparison, the binding of [3H]prazosin to rat BAT plasma membranes was inhibited by alpha1-adrenergic antagonists in the order of prazosin > WB 4101 = benoxathian > 5-methylurapidil. Although the order of the alpha1-adrenergic competition seemed to be rather typical for the alpha1B-adrenergic receptors, a molecular analysis on adrenoceptor mRNAs should be made to confirm the exact alpha1-adrenergic subtypes at the level of brown adipocytes, since the possibility of a mixture of different receptor subtypes in brown fat cells and/or tissue may interact with the pharmacological characterization. Thus, specific alpha1- and beta-adrenoceptor subtypes participate in the regulation of 5'DII activity in the rat brown adipocytes, and therefore, an impaired alpha1- and beta-adrenergic co-work may be involved in a defective BAT function, e.g., in obese Zucker rats, too. An interesting possibility is that the decreased number of alpha1-adrenoceptors in the BAT of obese Zucker rats is due to the decrease in the alpha1B-adrenoceptor subtype which would further be involved especially in the regulation of BAT 5'DII activity.     

Norepinephrine- and phorbol ester-induced phosphorylation of alpha(1a)-adrenergic receptors. Functional aspects

Maximal adrenergic responses in Rat-1 fibroblasts expressing alpha(1a)-adrenergic receptors are not blocked by activation of protein kinase C. In contrast, activation of protein kinase C induces the phosphorylation of alpha(1b)-adrenoreceptors and blocks their actions. The effect of norepinephrine and phorbol esters on alpha(1a)-adrenoreceptor phosphorylation and coupling to G proteins were studied. Both stimuli lead to dose-dependent receptor phosphorylation. Interestingly, protein kinase C activation affected to a much lesser extent the actions of alpha(1a)-adrenergic receptors than those of the alpha(1b) subtype (norepinephrine elicited increases in calcium in whole cells and [(35)S]GTPgammaS binding to membranes). Basal phosphorylation of alpha(1a)-adrenergic receptors was much less than that observed with the alpha(1b) subtype. The carboxyl terminus seems to be the main domain for receptor phosphorylation. Therefore, chimeric receptors, where the carboxyl-terminal tails of alpha(1a) and alpha(1b) adrenergic receptors were exchanged, were constructed and expressed. alpha(1a)-Adrenoreceptors wearing the carboxyl tail of the alpha(1b) subtype had a high basal phosphorylation and displayed a strong phosphorylation in response to norepinephrine and phorbol esters. Our results demonstrate that stimulation of alpha(1a)-adrenergic receptor, or activation of protein kinase C, leads to alpha(1a)-adrenergic receptor phosphorylation. alpha(1a)-Adrenoreceptors are affected to a much lesser extent than alpha(1b)-adrenoreceptors by protein kinase C activation.     

Interaction of the alpha(1B)-adrenergic receptor with gC1q-R, a multifunctional protein.

gC1q-R, a multifunctional protein, was found to bind with the carboxyl-terminal cytoplasmic domain of the alpha(1B)-adrenergic receptor (173 amino acids, amino acids 344-516) in a yeast two-hybrid screen of a cDNA library prepared from the rat liver. In a series of studies with deletion mutants in this region, the ten arginine-rich amino acids (amino acids 369-378) were identified as the site of interaction. The interaction was confirmed by specific co-immunoprecipitation of gC1q-R with full-length alpha(1B)-adrenergic receptors expressed on transfected COS-7 cells, as well as by fluorescence confocal laser scanning microscopy, which showed co-localization of these proteins in intact cells. Interestingly, the alpha(1B)-adrenergic receptors were exclusively localized to the region of the plasma membrane in COS-7 cells that expressed the alpha(1B)-adrenergic receptor alone, whereas gC1q-R was localized in the cytoplasm in COS-7 cells that expressed gC1q-R alone; however, in cells that co-expressed alpha(1B)-adrenergic receptors and gC1q-R, most of the alpha(1B)-adrenergic receptors were co-localized with gC1q-R in the intracellular region, and a remarkable down-regulation of receptor expression was observed. These observations suggest a new role for the previously identified complement regulatory molecule, gC1q-R, in regulating the cellular localization and expression of the alpha(1B)-adrenergic receptors.     

Activation and deactivation kinetics of alpha 2A- and alpha 2C-adrenergic receptor-activated G protein-activated inwardly rectifying K+ channel currents

Although G protein-coupled receptor-mediated signaling is one of the best studied biological events, little is known about the kinetics of these processes in intact cells. Experiments with neurons from alpha(2A)-adrenergic receptor knockout mice suggested that the alpha(2A)-receptor subtype inhibits neurotransmitter release with higher speed and at higher action potential frequencies than the alpha(2C)-adrenergic receptor. Here we investigated whether these functional differences between presynaptic alpha(2)-adrenergic receptor subtypes are the result of distinct signal transduction kinetics of these two receptors and their coupling to G proteins. alpha(2A)- and alpha(2C)-receptors were stably expressed in HEK293 cells at moderate ( approximately 2 pmol/mg) or high (17-24 pmol/mg) levels. Activation of G protein-activated inwardly rectifying K(+) (GIRK) channels was similar in extent and kinetics for alpha(2A)- and alpha(2C)-receptors at both expression levels. However, the two receptors differed significantly in their deactivation kinetics after removal of the agonist norepinephrine. alpha(2C)-Receptor-activated GIRK currents returned much more slowly to base line than did alpha(2A)-stimulated currents. This observation correlated with a higher affinity of norepinephrine at the murine alpha(2C)- than at the alpha(2A)-receptor subtype and may explain why alpha(2C)-adrenergic receptors are especially suited to control sympathetic neurotransmission at low action potential frequencies in contrast to the alpha(2A)-receptor subtype.     

Subtype-Selective Immunoprecipitation of the β2-Adrenergic Receptor

Most antibodies known to interact with beta-adrenergic receptors do not exhibit subtype selectivity, nor do they provide quantitative immunoprecipitation. A monoclonal antibody, G27.1 raised against a synthetic peptide corresponding to the C-terminus of the beta 2-adrenergic receptor of hamster, is selective for the beta 2 subtype. G27.1 provides nearly quantitative immunoprecipitation of the beta 2-adrenergic receptor from hamster lung that has been photoaffinity-labeled and solubilized with sodium dodecyl sulfate. Immunoprecipitation is completely blocked by nanomolar concentrations of the immunizing peptide. This antibody interacts with beta 2-adrenergic receptors from three rodent species, but not with those from humans. When C6 glioma cells, which contain both beta 1- and beta 2-adrenergic receptors, are photoaffinity-labeled in the absence or presence of subtype-selective antagonists, subtype-selective photoaffinity-labeling results. G27.1 can immunoprecipitate beta 2-, but not beta 1-, adrenergic receptors from these cells. Similar results were obtained following subtype-selective photoaffinity-labeling of membranes from rat cerebellum and cerebral cortex. The beta-adrenergic receptors from C6 glioma cells and rat cerebral cortex exist as a mixture of two molecular weight species. These species differ in glycosylation, as shown by endoglycosidase F digestion of crude and immunoprecipitated receptors.     

Site-directed anti-peptide antibodies define the topography of the beta-adrenergic receptor

Molecular cloning has revealed the primary structure of a number of G-protein-linked receptors. The organization and topography of these proteins predicted to have seven hydrophobic membrane-spanning domains, in contrast, have not been established. Antibodies were prepared against 11 peptides corresponding to each of the hydrophilic sequences of the hamster beta 2-adrenergic receptor. Each of the anti-peptide antibodies displayed immunoreactivity for its synthetic peptide antigen and beta 2-adrenergic receptor (Mr 65,000) on blots of cell membranes and of purified receptor. All but three anti-peptide antisera also displayed immunoreactivity toward human placental and rat fat cell beta 1-adrenergic receptors, reflecting the level of sequence identity that exists between the two subtypes, Chinese hamster ovary cells stably transfected with an expression vector harboring the cDNA encoding the hamster beta 2-adrenergic receptor provided a cell type with 2 million receptors/cell, suitable for in situ localization of the sequences used as antigens. Indirect immunofluorescence of intact and permeabilized cells performed with these site-directed anti-peptide antibodies permitted the assignment of the general topography of each of the hydrophilic sequences of this G-protein-linked receptor. The results support the predictive value of hydropathy analysis for one class of membrane proteins with multiple transmembrane-spanning domains.