THE JOURNAL OF BIOLOGICAL CHEMISTRY Y 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 14, Issue of May 15, pp. 6163%8189,199O Printed in U.S. A.

Molecular Cloning and Expression of the cDNA for a Novel CQ-Adrenergic Receptor Subtype*

(Received for publication, September 7, 1989)

Debra A. Schwinn$, Jon W. LomasneyQ, Wulfing Lorenzn, Pamela J. Szklut((, Robert T. Fremeau, Jr.**, Teresa L. Yang-Feng& Marc G. CaronlI))@, Robert J. LefkowitzT((Ul, and Susanna Cotecchiall From the Departments of $Anesthesiology, §Pathology, VMedicine, §§Cell Biology, WBiochemistry, **Neurobiology, and IlHoward Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710 and the $$Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510

A novel al-adrenergic receptor subtype has been cloned from a bovine brain cDNA library. The deduced amino acid sequence is that of a 466-residue polypep- tide. The structure is similar to that of the other adre- nergic receptors as well as the larger family of G protein-coupled receptors that have a presumed seven- membrane-spanning domain topography. The greatest sequence identity of this receptor protein is with the previously cloned hamster alB-adrenergic receptor being -72% within the presumed membrane-spanning domains. Localization on different human chromo- somes provides evidence that the bovine cDNA is dis- tinct from the hamster alB-adrenergic receptor. The bovine cDNA clone expressed in COS7 cells revealed lo-fold higher affinity for the al-adrenergic antago- nists WB4101 and phentolamine and the agonist oxy- metazoline as compared with the (YlB receptor, results similar to pharmacologic binding properties described for the alA receptor. Despite these similarities in phar- macological profiles, the bovine al-adrenergic receptor is sensitive to inhibition by the alkylating agent chlo- roethylclonidine unlike the alA-adrenergic receptor subtype. In addition, a lack of expression in tissues where the a1A subtype exists suggests that this receptor may actually represent a novel al-adrenergic receptor subtype not previously appreciated by pharmacologi- cal criteria.

Epinephrine and norepinephrine mediate their effects via binding to adrenergic receptors (aI, alp, &, p2) (1, 2). These receptors are encoded by different genes and are members of a much larger family of guanine nucleotide regulatory protein (G protein)-coupled receptors (3). Molecular cloning studies have revealed a growing heterogeneity of receptor subtypes. For example five different muscarinic cholinergic (4-8), three serotonergic (g-12), and two a*-adrenergic receptor subtypes (13, 14) have been recently identified by molecular cloning. Heterogeneity of al-adrenergic receptors (LY~* and alB sub-

* This work was supported in part by the American Society of Anesthesiologists/Association of University Anesthesiologists fellow- ship award, the Deutsche Forschungsgemeinschaft fellowship award, and by Grant 5R37-HL-16037 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 505426.

types) has been suggested by several pharmacological studies (15-19) based on differential sensitivity of al-adrenergic receptor-mediated responses to a variety of agonists (e.g. oxymetazoline) and antagonists (e.g. WB4101 and phentola- mine) as well as on different requirements of al-adrenergic receptor-induced responses for extracellular calcium (20).

Recently we reported the cloning of the cDNA which en- codes the hamster cul-adrenergic receptor purified from DDT1- MF, cells (21). The pharmacological properties of this CQ- adrenergic receptor resemble those described for the @B- receptor subtype. Here we present the cloning, sequencing, and expression of another oc,-adrenergic receptor subtype from bovine brain. This receptor shows the pharmacological properties proposed for the cYIA-adrenergic receptor subtype, but on the basis of a lack of expression in tissues where the alA subtype exists and its sensitivity to CEC’, it may actually represent a novel a!,-adrenergic receptor subtype not previ- ously appreciated by pharmacological criteria.

MATERIALS AND METHODS

Genomic and cDNA Library Screening-A human leukocyte ge- nomic library in EMBL3 (lo6 total recombinants, Clonetech, Palo Alto, CA) was screened with an oligonucleotide probe constructed from the hamster a,-adrenergic receptor cDNA (nucleotide 1028- 1094). Oligonucleotide probes were synthesized on an Applied Bio- systems 380B DNA synthesizer, purified on a 16% (w/v) denaturing polyacrylamide gel, and labeled with [Y-“~P]ATP at the 5’-hydroxyl group by phage T4 polynucleotide kinase. Duplicate filters were hybridized in 6 X SSC (1 x SSC = 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7), 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, 100 pg of sheared salmon sperm DNA/ml, lo6 cpm of “‘P-labeled probes/ml at 42 “C for 36 h. Filters were washed in 0.2 X SSC at 60 “C. A 0.32-kb PvuII/HindIII restriction fragment of the single genomic clone obtained was then used to screen a size-selected (2.0-4.4 kb) randomly primed bovine brain cDNA library in XZAP (300,000 total recombinants) generously provided by Dr. R. Dixon (Merck Sharp and Dohme) as described above. DNA fragments used as hybridization probes were labekd with [a-‘“P]dATP by random priming. Standard recombinant DNA and microbiological procedures were used (22).

DNA Sequencing-Nucleotide sequence analysis of both DNA strands was done by the Sanger dideoxy chain termination method (23) with overlapping restriction fragments and by primer extension in pTZ18R (Pharmacia LKB Biotechnology Inc.) or XZAP pBluescript SK with T7 DNA polymerase (Sequenase, United States Biochemical Corp., Cleveland, OH).

Expression-In order to construct an expression vector for the newly cloned bovine cDNA, we used the expression vector pBC12BI

1 The abbreviations used are: CEC, chloroethylclonidine; kb, kilo- base(s); bp, base pair(s); PCR, polymerase chain reaction; [“‘I] HEAT, 2-(~-(4-hydroxy-3-[‘25I]iodophenyl)ethylaminomethyl~-tetra- lone; AR, adrenergic receptor.

8183

by guest on April 24, 2020

http://ww

w.jbc.org/

Dow

nloaded from

8184 Cloning and Expression of a Novel aI-Adrenergic Receptor Subtype

(24) containing the human p2 cDNA (25) (pBCBI&). The pBCBI& was constructed as follows. After removing the pBC12BI NcoI site, the 2-kb NurI/EcoRI fragment of the human &adrenergic receptor was blunt-ended and ligated to the previously blunted HindIII/ BarnHI sites of the pBClSB1, thus obtaining the pBCBI& vector. The AJcoI/SalI restriction fragment of the pBCB& was then ligated to iVcoI/PstI (1.14 kb) and P&Sal1 (1.25 kb) restriction fragments from the cri-adrenergic bovine cDNA. This construct contained 40 base pairs (bp) of the 5’untranslated region of the human pz receptor adjacent to the Rous sarcoma virus promoter followed by 1398 bp of open reading frame and 967 bp of 3’-untranslated region of the bovine a,-adrenergic cDNA. This bovine cDNA construct was transfected into COS-7 cells by the DEAE-dextran method (24), and cells were harvested 72 h after transfection.

Ligand Binding-Transfected COS-7 cells from culture flasks (75 cm’) were scraped into 5 ml of TE solution (5 mM Tris.HCl, 5 mM EDTA, pH 7.4). A lysate was prepared with a Brinkmann homoge- nizer (model PT10/35, setting 5 for 8 s), the membranes were resus- pended in 50 mM Tris. HCl, 5 mM EDTA, 150 mM NaCl, pH 7.4, and quickly frozen and stored at -70 “C. Binding of the oli-adrenergic antagonist [‘z51]HEAT was measured as described (26) on 0.2 ml of diluted membrane aliquots (0.5-l gg of protein). For competition curves, a final [‘*‘I]HEAT concentration of 90 pM was used. For saturation curve analysis, [iZ51]HEAT concentrations ranged from 10 to 500 pM. Nonspecific binding was measured in the presence of 1 PM prazosin. Data were analyzed by computer using an iterative nonlin- ear regression program (27).

CEC Inactiuation-For CEC experiments, 100 pM CEC was incu- bated with transfected COS cell membrane lysates (50-100 fig of protein) or rat cortex membrane lysates (1 mg of protein) in-TE buffer at 37 “C for 20 min. The reaction was stotmed bv the addition of 4 ml of ice-cold TE solution and immediate centrifugation at 40,000 x g, 4 “C. Membranes were then washed three times with 5 ml of TE buffer prior to binding.

Northern Analysis-Total cellular RNA from various rat and bo- vine tissues, as well as COS7 cells transfected with either the hamster cum-adrenergic receptor cDNA or the bovine ai-adrenergic receptor cDNA, was isolated by the cesium chloride gradient method (28). For RNA blot analysis, poly(A)+-selected RNA was prepared using oligo(dT)-cellulose (29). Following denaturation by glyoxylation and fractionation by electrophoresis (30), RNA was immobilized on nylon membranes (Biotrans, ICN Corp.) and hybridized to 32P-labeled o(i- adrenergic receptor subtype cDNA probes.

Polymerase Chain Reaction-For DNA polymerase chain reaction (PCR) experiments, single strand cDNA was synthesized (31) using avian myeloblastoma virus reverse transcriptase (Promega), 100 pmol of random primer (d(N)s, Boehringer), and 5 pg of total cellular RNA from bovine tissues or 0.5 pg of total RNA from COS7 cells transfected with either the hamster cum-adrenergic receptor cDNA or the bovine cu,-adrenergic receptor cDNA. PCR amplification was performed (31) using this template or 30 ng of rat and bovine genomic DNA with oligonucleotide primers made from bovine oll-adrenergic receptor nucleotides 167-186 and the reverse complement of nucleotides 680- 701 (Fig. 1). PCR reactions were run for 30 cycles: 2 min at 92 “C, 2 min at 55 “C, and 3 min at 72 “C with Z’aq DNA polymerase (Perkin Elmer Cetus). PCR products were run on 2% agarose gels, transferred to nylon membranes (GeneScreen Plus, Du Pont-New England Nu- clear) and hybridized to 3ZP-labeled a,-adrenergic receptor subtype cDNA probes.

In Situ Hybridization-A 0.32-kb PuuII/HindIII fragment from the human homolog of the bovine ai-adrenergic was subcloned into pGEM-42 (Promega Biotec, Madison, WI). Adult human brain tissue was obtained from Dr. Edward Bird of the McLean Hospital Brain Bank, Belmont, MA. The dentate gyrus was dissected within 1 h of death and immersion-fixed in ice-cold 4% paraformaldehyde for 12 h. Coronol sections (15 mm) were cut on a cryostat and thaw-mounted onto poly-L-lysine-coated microscope slides. In situ hybridization using 35S-labeled single strand sense and antisense RNA probes (specific activity, >lO’ cpm/pg) was done as previously described (32, 33).

Drugs-Sources of drugs were as follows: [““IIHEAT (Du Pont- New England Nuclear); indoramin (gift from Abbott); methox- amine, phenylephrine, norepinephrine, epinephrine, oxymethazoline (Sigma); WB4101, p-aminoclonidine, chloroethylclonidine (Research Biochemicals Inc.); phentolamine (Ciba-Geigy); prazosin (Pfizer); yohimbine (Aldrich); idazoxan (Reckitt and Colman); and rauwol- seine (Roth, Karlsruhe, Federal Republic of Germany).

RESULTS

Screening of a human leukocyte genomic library with the labeled oligonucleotide probe constructed from the hamster cu,B-adrenergic receptor cDNA (nucleotides 1028-1094) iden- tified one clone under high stringency conditions (0.2 x SSC at 60 “C). This clone contained a 0.86-kb EcoRI/HindIII restriction fragment with an open reading frame between nucleotides 459 and 855. The open reading frame encoded a peptide where the first 43 residues were 67% identical with the hamster oci-adrenergic receptor putative seventh trans- membrane domain and the beginning of the carboxyl terminus (nucleotides 951-1077 of the hamster ai-adrenergic receptor) (21). Thereafter, the open reading frame diverged completely from the hamster ol,-adrenergic receptor sequence. In order to obtain a full-length clone, a size-selected (2.0-4.4 kb) bovine brain cDNA library was screened using as a probe a 0.32-kb PuuII/HindIII restriction fragment containing most of the open reading frame of the human genomic clone pre- viously obtained. A single clone with a 3.1-kb insert was isolated under high stringency conditions (0.2 x SSC at 60 “C). This bovine clone contained the same PuuII/HindIII restriction fragment as the human genomic clone. An initiator methionine at nucleotide 741 of the bovine cDNA clone started a 1398 bp open reading frame.

Fig. lA shows the restriction map of the bovine cDNA clone. Fig. 1B shows the bovine cDNA nucleotide and deduced amino acid sequence (open reading frame together with a short stretch of 5’untranslated sequence (96 bp) and the entire 3’-untranslated region (967 bp)). The open reading frame encodes a polypeptide of 466 amino acids with a cal- culated molecular mass of 51 kDa. Comparison of the deduced amino acid sequence of the bovine clone with that of previ- ously cloned G protein-coupled receptors revealed striking amino acid identity in the putative transmembrane domains of the various adrenergic receptors but most strikingly with the hamster cui-adrenergic receptor. The percentage identities in the putative transmembrane domains with each adrenergic receptor are the following: hamster (Yin, 72.1%; human (r&4, 43.2%; human (~~-C10, 41.5%; human /&, 43.2%; human pZ, 42.1% (13, 14, 21,25, 34,35). The level of amino acid identity within the putative transmembrane domains between the bovine cDNA clone and the hamster cylB-adrenergic receptor (72.1%) is similar to the level of amino acid identity within the transmembrane domains between the two a*-adrenergic receptor subtypes (75%) or between the ,6,- and &adrenergic receptor subtypes (75%). Comparison of the entire bovine cDNA-deduced amino acid sequence and the hamster (YlB- adrenergic receptor (Fig. 2, solid circles are identical amino acids) reveals that the NH* terminus (27% amino acid iden- tity), the COOH terminus (12% amino acid identity), and the third cytoplasmic loop (50% amino acid identity) represent the most divergent domains. These regions are also ones that differ the most in length and amino acid composition among other adrenergic receptors and G protein-coupled receptors. Since amino acid identity between hamster and human (YlB‘

adrenergic receptors is 99%,’ this suggests that the bovine clone does not encode the bovine homolog of the hamster alB‘

adrenergic receptor but rather a different oc,-adrenergic recep- tor subtype.

Three potential sites for N-linked glycosylation are present in the NH2 terminus (asparagine residues 7, 13, and 22). Several threonines and serines are present in the second and third cytoplasmic loops of this cDNA clone, representing potential sites for protein kinase C phosphorylation. A con-

’ D. A. Schwinn, P. J. Szklut, M. G. Caron, R. J. Lefkowitz, and S. Cotecchia, unpublished data.

by guest on April 24, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cloning and Expression of a Novel aI-Adrenergic Receptor Subtype 8185

sensus sequence for protein kinase A phosphorylation (amino acid residues 211-215) is present in the bovine a,-adrenergic receptor subtype cDNA in an analogous position to the con- sensus site seen in the hamster arls-adrenergic receptor cDNA. Phosphorylation has been observed as a mechanism of regu- lation for the ells-adrenergic receptor (37, 38). The presence of these consensus sequences suggests that this new cyl-adre- nergic receptor may also be regulated by phosphorylation.

To further confirm that the bovine cDNA obtained repre- sented a different gene product than the hamster LY~ -adrener- gic receptor, human somatic cell hybridization analysis was performed using as probes the 0.32-kb PuuII/HindIII frag- ment of the bovine cDNA and the 0.7-kb XhoI/BanHI frag- ment of the hamster cY1-adrenergic receptor. These studies showed that while the gene corresponding to the hamster aI- adrenergic receptor is located on human chromosome 5, the gene corresponding to the bovine cDNA is located on human chromosome K3

To further assess the functional identity of the cDNA isolated, a 2.4-kb fragment representing the coding region and the entire 3’-untranslated region was inserted into the expres- sion vector pBC12BI and used to transfect COS-7 cells. The COS-7 cells transfected with the vector containing the bovine cDNA were able to bind the al-adrenergic antagonist [lz51] HEAT with high specific activity (15 pmol/mg of protein) and with an affinity (60-70 PM) similar to that of the cloned hamster al=-adrenergic receptor. No binding activity was detected in untransfected COS-7 cells. Analysis of adrenergic agonist and antagonist competition curves showed the appro- priate pharmacology for al-adrenergic receptor binding. To compare ligand binding characteristics between the newly cloned bovine ol,-adrenergic receptor and the hamster (Yls- adrenergic receptor, competition curve analysis of agonists and antagonists for [lz51]HEAT binding was performed on membranes prepared from COS-7 cells transfected with the cDNA for either the bovine cY1-adrenergic receptor subtype or the hamster LulB-adrenergic receptor subtype. Comparison of the K, values for different compounds (Table I) reveals strik- ing differences between these two crl-adrenergic receptors. Specifically, the bovine al-adrenergic receptor subtype has more than lo-fold higher affinity for the (Y antagonists WB4101, phentolamine, corynanthine, and indoramin, as well as for the agonists oxymetazoline and methoxamine when compared with the hamster al-adrenergic receptor (Fig. 3). The LYE selective antagonist prazosin shows the same affinity for both cY,-adrenergic receptor subtypes, while epinephrine and norepinephrine are slightly more potent at the hamster culB-adrenergic receptor. Recently the existence of two (Ye- adrenergic receptor subtypes ((Y~* and (Y1s) has been suggested on the basis of differences in the affinities of the antagonists WB4101 and phentolamine and the agonist oxymetazoline for the two putative receptor subtypes (15-20), with the (YlA- adrenergic receptor subtype having lo-fold higher affinity for WB4101 than the (YIB. The lo-fold higher affinities of WB4101, phentolamine, and oxymetazoline for the bovine versus the hamster al-adrenergic receptor subtypes are in close agreement with those described for the n1A and alB- adrenergic receptor subtypes, respectively.

Ahother approach used to pharmacologically discriminate different al-adrenergic receptor subtypes has been the irre- versible inactivation of aI-adrenergic receptor binding by the alkylating derivative of clonidine, CEC (16, 17, 19). Minne- man and others (17,20) have shown that treatment with CEC inactivates 50-60% of the al-adrenergic receptors in rat cer- ebral cortex. Since the population of receptors left after CEC

3 T. L. Yang-Feng and U. Francke, manuscript in preparation.

by guest on April 24, 2020

http://ww

w.jbc.org/

Dow

nloaded from

8186 Cloning and Expression of a Novel q-Adrenergic Receptor Subtype

EXTRACELLULAR

amino terminus

INTRACELLULAR

FIG. 2. Seven-transmembrane-spanning model of the new bovine al-adrenergic receptor subtype showing amino acid identity with the hamster aIB-adrenergic receptor subtype. Solid circles indicate amino acids common to the corresponding position in the hamster ala-adrenergic receptor (21). Transmembrane domains are defined based on hydropathicity analysis (36). Potential N-linked glycosylation sites are shown as crosses.

inactivation shows only high affinity for WB4101, the (YlA- adrenergic receptor subtype has been suggested to be insen- sitive to CEC. To assess the effect of CEC on the bovine and hamster cY1-adrenergic receptors, transfected COS cell mem- branes were treated with 100 WM CEC for 20 min at 37 “C, and al-receptor ligand binding was measured with a saturating concentration of [lZ51]HEAT. CEC treatment inactivated 95 + 1 and 68 f 3% (mean + S.E., n = 3, p < 0.02) of the CQ binding in COS cells individually expressing the hamster (Y~B- and bovine al-adrenergic receptor, respectively. In rat cortex membranes, the same treatment with CEC inactivated 72 f 7.5% of al-adrenergic receptors, in agreement with the values previously reported (17, 19, 20). These results indicate that the bovine al-adrenergic receptor subtype is less sensitive to CEC inactivation than the hamster oils-adrenergic receptor subtype but more sensitive than has been suggested for the oclA-adrenergic receptor subtype (16, 17, 19).

In order to explore the tissue expression of the bovine al- adrenergic receptor and compare it with that of the hamster alB subtype, we performed Northern blot analysis on poly(A)+-selected mRNA of various rat and bovine tissues as well as utilized the DNA PCR with cDNA prepared from RNA of various bovine tissues. Northern blot analysis of rat mRNA has confirmed that the hamster al-adrenergic receptor previously cloned corresponds to the subtype described as the alB-receptor as indicated by strong hybridization to rat liver and cerebral cortex mRNA (15, 17, 19). However, no signal

was observed with the bovine (Ye subtype in any of the rat tissues (cerebral cortex, pituitary, hippocampus, brainstem, liver, heart, lung, kidney, spleen, aorta, adipose tissue, skeletal muscle, vas deferens) or bovine tissues (cerebral cortex, liver, heart, kidney, lung, adrenal) examined by Northern blot analysis or by PCR (data not shown). In situ hybridization of human dentate gyrus using antisense RNA probes derived from the human homolog of the bovine al-adrenergic receptor subtype revealed a restricted pattern of expression only in the granular cell layer (Fig. 4). These data suggest that the expres- sion of this receptor subtype is either very low or restricted to specific tissues or cellular populations.

DISCUSSION

We have cloned a cDNA encoding a novel al-adrenergic receptor subtype from bovine brain. Sequence homology with the previously cloned hamster alB-adrenergk? receptor (72.1% identity in the putative transmembrane domains) suggests the bovine cDNA is an al-adrenergic receptor. High affinity for the al-adrenergic antagonist [lz51]HEAT and overall rank order of potencies of agonists and antagonists confirms that the cDNA encodes an Lu,-adrenergic receptor. However, hu- man chromosome analysis provides evidence that the bovine oll-adrenergic receptor (localized to chromosome 8) is distinct from the hamster alB-adrenergic receptor (localized to chro- mosome 5). In addition, the homology between the bovine al- adrenergic receptor and the hamster ollB-adrenergic receptor

by guest on April 24, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cloning and Expression of a Novel aI-Adrenergic Receptor Subtype 8187

subtype in the putative transmembrane domains of the recep- tor is similar to that between az-adrenergic subtypes (75%) and @-adrenergic receptor subtypes (75%), suggesting that these receptors represent distinct cYl-adrenergic receptor sub- types. Ligand binding studies confirm that the bovine (Y,- adrenergic receptor subtype and the hamster ale-adrenergic

TABLE I Competition by agonists and antagonists for the binding of ?'"I]

HEAT to membranes prepared from COS-7 cells transfected with the pBC12BI expression vector containing the new bovine 01~ or the

hamster ala-adrenergic receptor cDNA Each concentration for competition curves was done in triplicate;

all isomers are (-) unless otherwise stated. Ratios (hamster (~is/ bovine (Ye) were rounded to the nearest digit. Results shown are the means of two experiments which agreed within 10%.

K,

BOVilX Hamster Ratio a1 am

Agonist Oxymetazoline p-Aminoclonidine Epinephrine Phenylephrine Norepinephrine (+)-Epinephrine Methoxamine Dopamine Serotonin Isoproterenol

Antagonist Prazosin WB4101 Phentolamine Indoramin Corynanthine Yohimbine Rauwolscine Idazoxan

nM

27 590

6,550 15,000 16,500 43,000 75,000

230,000 230,000

1,100,000

0.27 0.26 1 0.55 8.5 16 4.8 155 32 6 84 14

78 640 8 320 1,300 4

1,400 3,200 2 1,500 1,100 0.7

290 680

4,820 15,800 9,600

1,200,000 400,000 330,000

1,400,000

11

0.7 1 0.6

16 2 1

receptor subtype have clear pharmacological differences. Spe- cifically, the a-antagonists WB4101, phentolamine, corynan- thine, and indoramin, as well as agonists oxymetazoline and methoxamine, have IO-fold higher affinity for the bovine oi- adrenergic receptor subtype compared with the hamster CQ~- adrenergic receptor subtype. These data are in close agree- ment with ligand binding properties of the 0(iA- and LYE,- adrenergic receptors described in the literature, indicating that the bovine cDNA encodes a receptor with pharmacologic properties similar to the cYiA-adrenergic receptor subtype.

In contrast to the clear agreement between ligand binding properties reported for the cYiA-adrenergic receptor in the literature and those described for the bovine a,-adrenergic receptor subtype, CEC inactivation studies show some differ- ences. (Y~B- and alA-adrenergic receptors have been described as being sensitive or insensitive to the alkylating agent CEC, respectively (17, 19, 20). This classification originated from experiments in various tissues where CEC inactivation cor- related with a loss of low affinity sites for WB4lOl (corre- sponding to alB-adrenergic receptors), leaving only high affin- ity sites (corresponding to alA-adrenergic receptors). Cur data with transfected COS cell membranes treated with 100 PM CEC for 20 min at 37 “C show that the previously cloned (Y~B- adrenergic receptor subtype is in fact totally (95%) inactivated by CEC while the bovine Lu,-adrenergic receptor subtype is only partially (68%) inactivated. It is very difficult to conclude based on ligand binding studies with membranes derived from tissue homogenates containing mixtures of al-receptor sub- types whether the alA-receptor is completely insensitive to CEC or whether it is merely less sensitive than the (Yis. Although it seems clear that the arl-receptors remaining after CEC treatment in such preparations have ligand binding properties identical to those of the cloned bovine crl-adrener- gic receptor (e.g. high affinity for WB4101), it is also possible that some unspecified proportion of the al,+-receptor in the tissue homogenate was inactivated. Moreover, it is possible that another subtype of cq-adrenergic receptor exists with

-12.0 -11.0 -10.0 -9.0 -6.0 -7.0 -6.0 -5.0 -10.0 -9.0 -8.0 -7.0 -6.0 -5.0 -4.0 Log [WB4101] (M) Log [Oxymetazollnej (M)

FIG. 3. WB4101 (A) and oxymetazoline (B) competition for the binding of [‘*‘I]HEAT to membranes prepared from COS-7 cells transfected with the expression vector pBC12BL containing the cDNA for the new bovine al (circles) or the hamster cola-adrenergic (squares) receptor subtype. [‘251]HEAT was 90 pM, and 100% of [iZ51]HEAT bound was 10 PM. Results are representative of two experiments done in triplicate which agreed within 10%. The f(, estimates for these and other agonists and antagonists are listed in Table I.

by guest on April 24, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cloning and Expression of a Novel cul-Adrenergic Receptor Subtype

FIG. 4. In situ localization of the bovine aI-adrenergic receptor subtype hippocampus. A, x-ray autoradiographs of coronal section of the human dentate gyrus hybridized with the ?3- labeled antisense strand probe specific for the human homolog of the bovine cu,-adrenergic receptor. Specific labeling occurs over the granular cell layer. B, x-ray autoradiographs of serial sections hybridized with the 35S- labeled sense strand control probe resulted in only background labeling. C, emulsion autoradiographs (Kodak NTB2, hematoxylin and eosin) of the section shown in panel A magnified x 250. Specific hybridization to the granular cell layer (dense band of cells running through picture) is seen; pyramidal cells were not labeled. D, emulsion autoradiographs of the section shown in panel B magnified x 250 result in background labeling only.

similar pharmacological properties which may be completely insensitive to CEC inactivation.

In agreement with this hypothesis is the fact that while the pharmacological properties of the bovine cui-adrenergic recep- tor subtype suggest that it might represent the (YlA subtype, we have been unable to establish this identification by ob- serving mRNA species in tissues where the oclA-adrenergic receptor subtype has been previously described such as rat vas deferens and hippocampus. These observations strongly suggest that the bovine or,-adrenergic receptor represents a novel cYi-adrenergic receptor subtype and that expression of this receptor is quite specialized in either tissue distribution or developmental stage. In fact, even if at present we have not identified the bovine or rat tissues where the bovine CQAR subtype is expressed, in situ hybridization of human dentate gyrus slices reveals the presence of the human homolog of the bovine LQAR specifically in the granular cell layer. Recently the human and rat genes of a fifth muscarinic receptor have been cloned, but the expression of this receptor could not be detected in several rat tissues (8). These observations indicate that receptor heterogeneity might be more complex than predicted by pharmacological studies, and the isolation of different receptor subtypes by molecular cloning provides the most direct tool for the attribution of distinct receptor sub-

types to specific tissues. They also indicate the sensitivity and power of techniques such as PCR and in situ hybridization studies in determining the localization of new receptor sub- types. Since we have evidence for the presence of the gene analogous to the bovine alAR subtype in both the human and rat genome, a more extensive investigation by in situ hybrid- ization of human and rat tissues will be required to elucidate the tissue distribution as well as the level of expression of this novel alAR subtype.

The structural features of the new Lu,-receptor subtype are consistent with those of the other adrenergic receptors which have been cloned as well as with the wider family of G- protein-coupled receptors. The remarkable conservation of sequence within the presumed membrane-spanning domains between the two cri-receptor subtypes is consistent with their very similar ligand binding properties. This sequence conser- vation extends to those regions of the third cytoplasmic loop and carboxyl-terminal cytoplasmic tail which are presumed to lie closest to the plasma membrane. Inasmuch as these regions of the receptor molecule have been suggested to be those responsible for coupling to G-proteins, this might sug- gest that the effector function of these receptor subtypes might also be similar. However it has been speculated that al-receptor subtypes might mediate distinct functions (19).

by guest on April 24, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Cloning and Expression of a Novel cq-Adrenergic Receptor Subtype

The availability of clones for distinct Lul-adrenergic receptor 16. subtypes should now facilitate refinement not only of the structural basis of adrenergic receptor ligand binding but also

17 ’

of the signal transduction mechanisms utilized by different 18. aI-adrenergic receptor subtypes.

8189

Han, C., Abel, P. W. & Minneman, K. P. (1987) Mol. Pharmacol. 32,505-510

Minneman, K. P., Han, C. & Abel, P. W. (1988) Mol. Pharmacol. 33,509-514

Hieble, J. P., Mattherw, W. D., DeMarinis, R. M. & Ruffolo, R. R. (1987) in The Alpha-l Adrenergic Receptors (Ruffolo, R. R., ed) pp. 325-343, Humana Press, Clifton, NJ

Minneman, K. P. (1988) Pharmacol. Reu. 40,87-119 Han, C., Abel, P. W. & Minneman, K. P. (1987) Nature 329,

333-335

Acknowledgments-We gratefully acknowledge Sabrina Exum for 19.

assistance with tissue culture and ligand binding, K. Theisen for 2O

’ oligonucleotide synthesis, Dr. R. Dixon (Merck Sharp and Dohme) for generously providing the bovine brain cDNA library in XZAP, and

21 ’

Drs. John Regan and Sheila Collins for helpful discussions.

REFERENCES 22.

1. Ahlquist, R. P. (1948) Am. J. Physiol. 153, 586-600 2. Lefkowitz. R. J. & Caron. M. G. (1988) J. Biol. Chem. 263.4993-

4996 3. Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615-649 4. Kubo, T., Fukuda, K., Mikami, A., Maeda, A., Takahashi, H.,

Mishina, M., Haga, T., Ichiyama, A., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T. & Numa, S. (1986) Nature 323, 411-416

5. Kubo, T., Maeda, A., Sugimoto, K., Akiba, I., Mikami, A., Taka- hashi, H., Haga, T., Haga, K., Ichiyama, A., Kangawa, K., Matsuo, H., Hirose, T. & Numa, S. (1986) FEBS Lett. 209, 367-372

6. Peralta, E. G., Winslow, J. W., Peterson, G. L., Smith, D. H., Ashkenazi, A., Ramachandran, J., Schimerlik, M. I. & Capon, D. J. (1987) Science 236,600-605

7. Bonner, T. I., Buckley, N. J., Young, A. C. & Brann, M. R. (1987) Science 237, 527-532

8. Bonner, T. I., Young, A. C., Brann, M. R. & Buckley, N. J. (1988) Neuron 1,403-410

9. Fargin, A., Raymond, J. R., Lohse, M. J., Kobilka, B. K., Caron, M. G. & Lefkowitz, R. J. (1988) Nature 335, 358-360

10. Kobilka, B. K., Frielle, T., Collins, S., Yang-Feng, T., Kobilka, T. S., Francke, U., Lefkowitz, R. J. & Caron, M. G. (1987) Nature 329, 75-79

11. Julius, D., MacDermott, A. B., Axel, R. & Jessel, T. M. (1988) Science 241,558-564

12. Pritchett, D. B., Bach, A. W. J., Wozny, M., Taleb, O., Dal Taso, R., Shih, J. C. & Seeburg, P. H. (1988) EMBOJ. 7, 4135-4140

13. Kobilka, B. K., Matsui, H., Kobilka, T. S., Yang-Feng, T. L., Francke, U., Caron, M. G., Lefkowitz, R. J. & Regan, J. W. (1987) Science 238,650-656

14. Regan, J. W., Kobilka, T. S., Yang-Feng, T. L., Caron, M. G. Lefkowitz, R. J. & Kobilka, B. K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6301-6305

15. Morrow, A. L. & Creese, I. (1986) Mol. Pharmacol. 29, 321-330

23.

24. 25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36. 37.

38.

Cotecchia, S., Schwinn, D. A., Randall, R. R., Lefkowitz, R. J., Caron, M. G. & Kobilka, B. K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7159-7163

Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467

Cullen, B. R. (1987) Methods Enzymol. 152, 684-704 Kobilka, B. K., Dixon, R. A. F., Frielle, T., Dohlman, H. G.,

Bolanowski, M. A., Sigal, I. S., Yang-Feng, T. L., Francke, U., Caron, M. G. & Lefkowitz, R. J. (1987) Proc. N&l. Acad. Sci. U. S. A. 84, 46-50

Cotecchia, S., Leeb-Lundberg, L. M. F., Hagen, P-O., Lefkowitz, R. J. & Caron, M. G. (1986) Life Sci. 37, 2389-2398

DeLean, A., Hancock, A. A. & Lefiowitz, R. J. (1982) Mol. Pharmacol. 21, 5-16

Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. (1979) Biochemistry 18, 5294-5299

Aviv, H., & Leder, P. (1972) Proc. Natl. Acad. Sci. ZJ. S. A. 69, 1408-1412

Collins, S., Caron, M. G. & Lefkowitz, R. J. (1988) J. Biol. Chem. 263,9067-9070

Kawasaki, E. S. & Wang, A. M. (1989) in PCR Technology: Principles and Applications for DNA Amplification (Erlich, H. A., ed) Stockton Press, New York

Fremeau, R. T., Jr., Lundblad, J. R., Pritchett, D. B., Wilcox, J. N. & Roberts, J. L. (1986) Science 234, 1265-1269

Fremeau, R. T., Jr., Autelitano, D. J., Blum, M., Wilcox, J. & Roberts, J. L. (1989) Mol. Brain Rex 6, 197-201

Frielle, T., Collins, S., Daniel, K. W., Caron, M. G., Lefkowitz, R. J. & Kobilka, B. K. (1987) Proc. N&l. Acad. Sci. U. S. A. 84, 7920-7924

Dixon, R. A. F., Kobilka, B. K., Strader, D. J., Benovic, J. L., Dohlman, H. G., Frielle, T., Bolanowski, M. A., Bennett, C. D., Rands, E., Diehl, R. E., Mumford, R. A., Slater, E. E., Sigal, I. S., Caron, M. G., Lefkowitz, R. J. & Strader, C. D. (1986) Nature 321,75-79

Kyte, J. & Doolittle, F. F. (1982) J. Mol. Biol. 157, 105-132 Leeb-Lundberg, L. M. F., Cotecchia, S., DeBlasi, A., Caron, M.

G. & Lefiowitz, R. J. (1987) J. Biol. Chem. 262, 3098-3105 Bouvier, M., Leeb-Lundberg, L. M. F., Benovic, J. L., Caron, M.

G. & Lefkowitz, R. J. (1987) J. Biol. Chem. 262,3106-3113

by guest on April 24, 2020

http://ww

w.jbc.org/

Dow

nloaded from

M G Caron, R J Lefkowitz and S CotecchiaD A Schwinn, J W Lomasney, W Lorenz, P J Szklut, R T Fremeau, Jr, T L Yang-Feng,

receptor subtype.Molecular cloning and expression of the cDNA for a novel alpha 1-adrenergic

1990, 265:8183-8189.J. Biol. Chem. 

  http://www.jbc.org/content/265/14/8183Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/265/14/8183.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on April 24, 2020

http://ww

w.jbc.org/

Dow

nloaded from