1. Receptor Mechanisms Structure and Molecular Biology of Transmitter Receptors 1 J. CRAIG VENTER and CLAIRE M. FRASER

Introduction The neurotransmitter receptors of the autonomic nervous system play important roles in the modulation of physiologic function in the airways and other organ systems. Cloning and sequence analysis of genes encoding the receptors have revealed that these pharmacologically diverse classes of receptors, which interact with guanine nucleotide regulatory proteins, are members of a large supergene family and share significant structural homology at both the DNA and protein levels. In this review, we will outline recent work on the structure and molecular biology of alpha-adrenergic and muscarinic cholinergic receptors. Recently, we reviewed the advances in our understanding of the structure, function, and molecular biology of beta-adrenergic receptors (I), and that review together with this summary serve to outline the current knowledge in this area. Based on monoclonal antibody cross-reactivities and limited proteolysis studies, we proposed in the early 1980sthat alpha-adrenergic and muscarinic cholinergic receptors were members of a family of receptors that interacted with common membrane effector molecules (2, 3). In similar studies, Shreeve and coworkers (4) demonstrated monoclonal antibody cross-reactivity between alpha.- and alpha.-adrenergic receptors leading us to propose that receptor subclasses may have arisen as gene duplication events (3, 4). The application of molecular biology to the study of neurotransmitter receptors has led to substantial advances in our understanding of receptor structure and function (5). Cloning and sequence analysis of genes encoding receptors have confirmed our earlier observations of the homology between receptor classes and of the existence of a supergene family of receptors, which includes adrenergic (6-10), muscarinic cholinergic (6, 11-13), serotonin (14, 15),dopamine (16), angiotensin (17), substance K (18), and yeast mating factor receptors (19), as well as the visual pigments, the opsins (20-22), and bacteriorhodopsin (23). Site-directed mutagenesis studies have begun to identify important amino acid residues involved in ligand binding and receptor activation and provide a means for elucidation of the molecular basis of signal transduction across membranes. Cloning and Sequence Analysis of Alpha-adrenergic Receptor Genes Alpha-adrenergic receptors that bind catecholamines were originally subclassified into alpha,- and alpha.-adrenergic subtypes based

SUMMARY Details of receptor structure and function that were unavailable as recently as two years ago are now readily obtainable through the application of molecular biological techniques. Cloning and sequence analysis of neurotransmitter receptor genes have provided information on the primary structure of these proteins, revealing the relationship between pharmacologically diverse families of receptors. Knowledge of the primary structure of receptors has allowed for prediction of secondary structure and the construction of three-dImensional models. Permanent expression of cloned neurotransmitter receptor genes In cultured cells is providing unlimited sources of pure receptor, which allows for pharmacological and biochemical studies on single receptor subtypes. The use of site-directed mutagenesis to elucidate the relationship between protein structure and function has provided considerable Information on the role of certain conserved amino acids In receptor function and has suggested possible molecular mechanisms of signal transduction across membranes. The article will review some of these recent developments in the area of neurotransmitter receptors and point out the utility of molecular biology In these endeavors. AM REV RESPIR DIS 1990; 141:S99-S105

on anatomical and pharmacologic criteria (24, 25). Alpha.- and alpha.-adrenergic receptors are coupled to at least two distinct second messenger pathways through G proteins: alpha.adrenergic receptors stimulate breakdown of phosphoinositides and alphas-adrenergic receptors inhibit adenylate cyclase activity. The alpha-adrenergic receptor has been implicated in a number of physiologic functions including smooth muscle contraction and regulation of hepatic glycogen metabolism, whereas the alphaj-adrenergic receptor appears to play an important role in platelet aggregation, regulation of insulin release from the pancreas, and regulation of lipid metabolism in adipose tissue. During the past two years the genes encoding a hamster alpha-adrenergic receptor (10) and two distinct human alpha-adrenergic receptors from platelets (7, 26) and the kidney (27) have been cloned and sequenced. As illustrated in figure 1, these receptors share considerable homology among themselves and with other receptors in the supergene family, such as beta-adrenergic and muscarinic cholinergic receptors. The hamster alpha.adrenergic receptor gene encodes a protein of 515 amino acids with a molecular mass of 56,000 (10), a value in close agreement with the apparent molecular mass of the deglycosylated alpha.sadrenergic receptor (28). The platelet (7, 26) and kidney (27) alphaj-adrenergic receptors encode proteins of 450 and 461 amino acids, respectively. As observed for other receptors in this family, the alpha.- and alphas-adrenergic receptors contain sites for N-linked glycosylation (figure 1). The primary sequences of the alpha-adrenergic receptors contain seven stretches of hydrophobic amino acids, which are large enough to span the lipid bilayer and presumably represent transmem-

brane helices in these proteins (figure 1). Interestingly, the regions of highest amino acid homology among the neurotransmitter receptors lie within the putative transmembrane spanning domains suggesting that these regions of the receptors are critical for normal receptor structure and/or function.

Expression of Alpha-adrenergic Receptors in Cultured Cells In order to confirm the identity of the proteins encoded by the cloned genes and to characterize their pharmacologic and biochemical properties it is necessary to utilize systems for the expression of these proteins in mammalian cells. To date, the hamster alpha.-adrenergic receptor has been transiently expressed in COS-7 cells in culture, has been shown to bind alpha-adrenergic ligands with the expected order of potency for an alpha.adrenergic receptor, and to couple to inositol phospholipid metabolism (10). More detailed characterization of the alpha.-adrenergic receptor has been precluded by the transient expression system employed. In contrast to the data with the alpha. receptor, we have utilized plasmid expression vectors to achieve permanent expression of neurotransmitter receptors in cultured Chinese hamster ovary (CRO) cells that lack endogenous receptors and have performed detailed pharmacologic and biochemical characterization of the human alphas-adrenergic receptor from the platelet (26). As illustrated in figure 2, the alpha, receptor expressed in CRO , From the Section of Receptor Biochemistry and Molecular Biology, Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland.

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cells displayed pharmacology characteristic of an alpha.A receptor subtype (29) with a high affinity for yohimbine (Kd = 1 nM) and a low affinity for prazosin (Kd = 10,000 nM). Agonists displayed a rank order of potency in ligand-binding assays of paraaminoclonidine ~ UK 14303 >(- j-epinephrine >(- )-norepinephrine >(- )-isoproterenol, consistent with the identification of this protein as an alpha, receptor (figure 2). The role of the alpha, receptor in modulation of intracellular cAMP production was investigated in transfected CHO cells (figure 3). At low epinephrine concentrations (l to 100 nM), epinephrine attenuated forskolinstimulated cAMP accumulation by up to 60070

in a receptor density-dependent manner. At higher epinephrine concentrations, cAMP levels were increased to 140% of the forskolinstimulated level. Pertussis toxin pretreatment of cells eliminated alpha, receptor-mediated attenuation of forskolin-stimulated cAMP levels and enhanced the receptor density potentiation of forskolin-stimulated cAMP concentrations from three- to eightfold (figure 3). The nonselective phospholipase A2 inhibitor, quinacrine, antagonized the receptor-mediated stimulation of cAMP production in pertussis toxin-treated cells in a dose-dependent manner (figure 4). Cells treated with quinacrine in the absence of pertussis toxin displayed an epinephrine-mediated inhibition

of forskolin-stimulated cAMP production of up to 90% with no increases in cAMP at high epinephrine concentrations (figure 5). These data suggest that the human alphas-adrenergic receptor in CHO cells may simultaneously couple to more than one effector, including. a pertussis toxin-sensitive attenuation of adenylate cyclase and a pertussis toxin-insensitive pathway that results in potentiation of intracellular cAMP levels (26).

Cloning and Sequence Analysis of Muscarinic Cholinergic Receptor Genes Muscarinic cholinergic receptors have a distinct pharmacology and modulate a large ar-

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produced a dose-dependent increase in the hydrolysisof phosphoinositides (PI). Pertussis toxin reduced carbachol-stimulated PI turnover, suggesting that this muscarinic receptor is coupled to the hydrolysis of inositol

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Fig. 6. Comparison of the deduced amino acid sequences of the rat muscarinic cholinergic receptors. The M2 receptor sequence is from reference 6 and the Ml, m3, and m4 sequences are from reference 36. The sequences were aligned with gaps introduced to display maximum homology by the GAP program. The amino acids conserved in all four proteins are shown below the sequences, indicated by the letter for that amino acid or by an asterisk if favored substitutions are present. Charged groups, cysteines, and prolines that are conserved in all four proteins are shown above the sequences. Putative membrane spanning regions are indicated by hatched bars. (From Kerlavage and coworkers (30) with permission from the pubushet)

lipids via a pertussis toxin-sensitive G protein. At concentrations of carbachol up to 10 mM, no stimulatory or inhibitory effect on basal or PGEt·stimulated cAMP formation in B-82 cells was observed (39). In contrast to the results obtained from expression studies of the Ml muscarinic receptor were those of Ashkenazi and colleagues (41) on the coupling of a porcine M2 mus-

Fig. 7. Helical wheel representation of rat Ml muscarinic receptor transmembrane segments. Single letter amino acid codes are shown. All transmembrane segments are modeled as alpha-helices. Shaded regions indicate the moet hydrophobic face of each helix. Odd-numbered helical wheels start at the extracellular surface and proceed clockwise around the wheel with each successive residue five spokes removed from the previous. Evennumbered helices begin at the intracellular surface and proceed counterdockwise around the wheel.

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MOLECULAR BIOLOGY OF RECEPTORS

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carinic cholinergic receptor to cellular effectors in CHO cells. The muscarinic agonist, carbachol, both inhibited adenylate cyclase and stimulated PI hydrolysis in transfected cells. The stimulation of PI hydrolysis was significantly less efficient and more depen-

dent on high levels of receptor expression than inhibition of adenylate cyclase. Although these data suggest that one muscarinic receptor subtype may couple to multiple effector systems, the physiologic relevance of M2 receptor stimulation of PI hydrolysis is ques-

tionable. Additional studies on the function of other genetically defined muscarinic receptor subtypes will aid in elucidating the nature of receptor effector coupling.

Site-directed Mutagenesis of M1 Muscarinic Cholinergic Receptors Most members of the neurotransmitter receptor gene family contain highly conserved aspartic acid (Asp) residues in the second and third transmembrane helices (figure 1). Sitedirected mutagenesis of these residues in the human betas-adrenergic receptor and in bacteriorhodopsin has demonstrated that these aspartic acids are involved in ligand binding and receptor activation by agonists and proton translocation, respectively (42-45), suggesting an important role for these amino acids in receptor function. In order to investigate the role of the conserved aspartic acids in muscarinic receptor function, we made single point mutations in the Ml muscarinic receptor gene to independently convert Asp 71 in the second transmembrane domain and Asp!" and Asp l22 in the third transmembrane domain to asparagine (Asn) and then permanently transfected CHO cells with each mutant gene. Mutation of Asp" produced a mutant receptor that had normal antagonist and partial agonist binding and a U.5-fold higher affinity for carbachol than the wild-type receptor (figure 9). These data are similar to those derived from site-directed mutagenesis of Asp 79 in the betas-adrenergic receptor (42) and suggest that different coordinates may exist for the binding of agonists and antagonists in these receptor classes. Unlike the corresponding mutation in the beta. receptor, which decreased agonist affinity, substitution of Asp 71 with Asn in the muscarinic receptor increased agonist affinity. These data support a role for the conserved Asp in agonist binding in both beta-adrenergic and muscarinic receptors; however, the opposing changes in receptor affinity suggest that the nature of agonist binding to these receptors may be fundamentally different. Asn" mutant muscarinic receptors induced only minimal stimulation of PI hydrolysis in transfected cells (figure 10), similar to the findings with the Asn 79 mutant beta receptor that was unable to mediate agonist stimulation of adenylate cyclase (42). These data suggest that the aspartic acid residue in the second transmembrane domain of all members in this gene family may play a common role in agonist activation of intracellular effectors. Substitution of Asp 122 with Asn, in the third transmembrane domain of the muscarinic receptor, produced a mutant receptor that had normal antagonist and partial agonist affinity but a 3.2-fold higher affinity for carbachol (figure 9). Both carbachol and oxotremorine were able to stimulate PI hydrolysis in cells expressing the Asn 122 receptor. While maximal levels of PI hydrolysis were observed in these cells, the EC•• for carbachol stimulation of PI hydrolysis was shifted from 3 11M

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Fig. 9. Muscariniccholinergicagonist bindingto wild-type and mutant M1 muscarinic receptors. Membranesfrom CHO cells expressing wildtype, Asn71 or Asn' 22 mutant M1 receptors were incubated with a Kd concentration of ['H]ONB and the indicated concentrations of carbachol or oxotremorinefor 60 min at 37" C. Samples were filtered and counted for specific ONB binding. Open circles, squares, and triangles represent ONB binding to wildtype, Asn71, and Asn'22 mutant receptors, respectively, in the presence of oxotremorine. The corresponding solidsymbolsrepresentONB bindingto the indicatedreceptorsinthe presence ofcarbachol.(Datafrom reference40.)

with the wild-type receptor to 30 ~M with the Asn 122 mutant receptor (figure 10). The discrepancy between the increase in receptor affinity for carbachol and the decreased efficacy of full and partial agonists to elicit maximal responses suggest a change in the efficiency of receptor coupling to G protein,s. Substitution of Asp,05 with Asn produced a mutant receptor with decreased affinity for agonists and antagonists, such that receptor detection with radioligand binding assays was limited. These findings are similar to those from mutagenesis of the corresponding aspartic acid residue in the hamster betas-adrenergic receptor (44) and implicate this amino acid in ligand binding either directly or indirectly in the maintenance of a receptor conformation required for high affinity ligand binding.

Conclusions The application of molecular biology to the study of neurotransmitter receptor structure and function has provided some insights heretofore not possible with pharmacologic and biochemical assays. These new technologies now allow for the detection of receptors at the protein, RNA, and DNA levelsand will be of tremendous utility in elucidating the molecular basis of signaltransduction and the mechanisms of receptor regulation. References 1. Fraser CM, Venter JC. Beta-adrenergic receptors: relationship of primary structure, receptor function, and regulation. Am Rev Respir Dis 1990; 141(Suppl:S22-30). 2. Venter JC. Muscarinic cholinergic receptor. Receptor size, membrane orientation and absence of major phylogenetic structural diversity. J Biol Chern 1983; 258:4842-8. 3. Venter JC, Eddy B, Hall LM, Fraser CM. Monoclonal antibodies detect the conservation of muscarinic cholinergic receptor structure from Drosophila to human brain and detect possible structural homology with alphal-adrenergic receptors. Proc Nat! Acad Sci USA 1984; 81:272-6.

4. Shreeve SM, Fraser CM, Venter JC. Molecular comparison of alphal and alpha2-adrenergic receptors suggests that these proteins are structurally related "isoreceptors." Proc Nat! Acad Sci USA 1985; 82:4842-6. 5. Venter JC, Fraser CM, Kerlavage AR, Buck MA. Molecular biology of adrenergic and muscarinic cholinergic receptors: A perspective. Biochem Pharmacol 1989; 38:1197-208. 6. Gocayne JD, Robinson DA, FitzGerald MG, et al. Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: Further evidence for a multigene family. Proc Nat! Acad Sci USA 1987; 84:8296-300. 7. Kobilka BK, Maysui H, Kobilka TS, et al. Cloning, sequencing and expression of the gene coding for the human platelet alpha2-adrenergic receptor. Science 1987; 238:650-6. 8. Frielle T, Collins S, Daniel KW, Caron MG, Lefkowitz RJ, Kobilka B. Cloning of the eDNA for the human betal-adrenergic receptor. Proc Nat! Acad Sci USA 1987; 84:7920-4. 9. Chung F-Z, Lentes K-U, Gocayne J, et al. Cloning and sequence analysis of the human brain betaadrenergic receptor. Evolutionary relationship to rodent and avian beta-receptors and porcine muscarinic receptors. FEBS Lett 1987; 211:200-6. 10. Cotecchia S, Schwinn DA, Randall RR, Lefkowitz RJ, Caron MG, Kobilka BK. Molecular cloning and expression of the eDNA for the hamster alphal-adrenergic receptor. Proc Nat! Acad Sci USA 1988; 85:7159-63. 11. Kubo T, Maeda A, Sugimoto K, et al. Primary structure of porcine cardiac muscarinic acetylcholine receptor deduced from the eDNA sequence. FEBS Lett 1986; 299:367-72. 12. Kubo T, Fukuda K, Mikami A, et al. Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor. Nature 1986; 323:411-6. 13. Peralta EG, Ashkenazi A, Winslow JW, Smith DH, Ramachandran J, Capon DJ. Distinct primary structures, ligand-binding properties and tissue specific expression of four human muscarinic acetylcholine receptors. EMBO J 1987; 6:3923-9. 14. Julius D, MacDermott AB, Axel R, Jessell TM. Molecular characterization of a functional cDNA encoding the serotonin lc receptor. Science 1988; 241:558-64.

Fig. 10. Measurement of phosphoinositideturnover in CHO cells transfected with wild-type and mutant M1 muscarinicreceptors. CHOcells containingequivalent densities ofwild-type, Asn71 or Asn'22 mutant receptors were assayed for carbachol- and oxotremorine-stimulated phosphoinositide hydrolysis accordingto the method of Berridge. Data are expressed as foldbasal stimulationof phosphoinositide hydrolysis, which is directly proportional to the extent of lipid labeling by radiolabeled inositol. (Data from reference 40.) 15. Fargin A, Raymond JR, Lohse MJ, Kobilka BK, Caron MG, Lefkowitz RJ. The genomic clone G-21 which resembles a beta-adrenergic receptor sequence encodes the 5-HTlA receptor. Nature 1988; 335:358-60. 16. Bunzow JR, Van Tol HHM, Grandy DK, et al. Cloning and expression of a rat D2 dopamine receptor eDNA. Nature 1988; 336:783-7. 17. Jackson TR, Blair LAC, Marshall J, Goedert M, Hanley M. The mas oncogene encodes an angiotensin receptor. Nature 1988; 335:437-40. 18. Masu Y, Nakayama K, Tamaki H, Harada Y, Kuno M, Nakanishi S. eDNA cloning of bovine substance K receptor through oocyte expression system. Nature 1987; 329:836-8. 19. Marsh L, Herskowitz I. STE2 protein of Saccharomyces kiuyveri is a member of the rhodopsin/beta-adrenergic receptor family and is responsible for recognition of the peptide ligand alpha factor. Proc Nat! Acad Sci USA 1988; 85:3855-9. 20. Nathans J, Hogness DS. Isolation, sequence analysis and intron-exon arrangement of the gene encoding bovine rhodopsin. Cell 1983; 34:807-14. 21. Nathans J, Hogness DS. Isolation and nucleotide sequence of the gene encoding human rhodopsin. Proc Nat! Acad Sci USA 1984; 81:4851-5. 22. Zuker CS, Cowman AF, Rubin GM. Isolation and structure of a rhodopsin gene from D. melanogaster. Cell 1985; 40:851-8. 23. Martin RL, Wood C, Baehr W, Applebury ML. Visual pigment homologies revealed by DNA hybridization. Science 1986; 232:1266-9. 24. Langer SZ. Presynaptic regulation of catecholamine release. Biochem Pharmacol 1974; 23: 1793-800. 25. Starke K. Alpha-adrenoceptor subclassification. Rev Physiol Biochem Pharmacol 1981; 88: 199-236. 26. Fraser CM, Arakawa S, McCombie WR, Venter JC. Cloning, sequence analysis and permanent expression of a human alpha2-adrenergic receptor in CHO cells: evidence for independent pathways of receptor coupling to adenylate cyclase attenuation and activation. J Bioi Chem 1989;264:11754-61. 27. Regan JW, Kobilka TS, Yang-Feng TL, et al. Cloning and expression of a human kidney eDNA for an alpha2-adrenergic receptor subtype. Proc Nat! Acad Sci USA 1988; 85:6301-5. 28. Sawutz DG, Lanier SM, Warren CD, Graham RM. Glycosylation of the mammalian alphal-

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adrenergic receptor by complex type N-linked oligosaccharides. Mol Pharmacol1987; 32:565-71. 29. Bylund DB. Heterogeneity of alpha2-adrenergic receptors. Pharmacol Biochem Behav 1985;22: 835-43. 30. Kerlavage AR, Fraser CM, Venter JC. Muscarinic cholinergic receptor structure: molecular biological support for subtypes. Trends Pharmacol Sci 1987; 8:426-31. 31. Hammer R, BerrieCP, Birdsall NJM, Burgen ASV, Hulme EC. Pirenzepine distinguishes between different subclasses of muscarinic receptors. Nature 1980; 283:90-2. 32. Birdsall NJM, Hulme EC. Muscarinic receptor subclasses. Trends Pharmacol Sci 1983;4: 454-63. 33. Hirschowitz BI, Hammer R, Giachetti A, Keirns J J, Levine RR, eds. Trends Pharmacol Sci 1984; 5(Suppl). 34. Brown JH, Brown SL. Agonists differentiate muscarinic receptors that inhibit cyclic AMP formation from those that stimulate phosphoinositide metabolism. J Bioi Chern 1984; 259:3777-81. 35. Peralta EG, Winslow JW, Peterson GL, et at. Primary structure and biochemical properties of

an M2 muscarinic receptor.Science1987; 236:600-5. 36. Bonner 'I'l, Buckley NJ, Young AC, Brann MR. Identification of a family of muscarinic acetylcholine receptor genes. Science 1987; 237:527-32. 37. Henderson R, Unwin PTN. Three dimensional model of purple membrane obtained by electron microscopy. Nature 1975; 257:28-32. 38. Lai J. Mei L, Roeske WR, Chung F-Z, Yamamura HI, Venter JC. The cloned murine Ml muscarinic receptor is associated with the hydrolysis of phosphatidylinositols in transfected murine B82 cells. Life Sci 1988; 42:2489-502. 39. Mei L, Lai J. Roeske WR, Fraser CM, Venter JC, Yamamura, HI. Pharmacological characterization of the Ml muscarinic receptors expressed in murine fibroblast B82 cells. J Pharmacol Exp Ther 1989 (In Press). 40. Fraser CM, Wang C-D, Robinson DA, Gocayne JD, Venter JC. Site-directed mutagenesis of M, muscarinic acetylcholine receptors: conserved aspartic acids play important roles in receptor function. Mol Pharmacol 1989; 36:840-7. 41. Ashkenazi A, Winslow JW, Peralta EG, et at. An M2 muscarinic receptor subtype coupled to both

adenylyl cyclase and phosphoinositide turnover. Science 1987; 238:672-5. 42. Chung F-Z, Wang C-D, Potter PC, Venter JC, Fraser CM. Site-directed mutagenesis and continuous expression of human beta-adrenergic receptors. Identification of a conserved aspartate residue involved in agonist binding and receptor activation. J Bioi Chern 1988; 263:4052-5. 43. Fraser CM, Chung F-Z, Wang C-D. VenterJC. Site-directedmutagenesis of human beta-adrenergic receptors: Substitution of aspartic acid 130 by asparagine produces a receptor with high affinity agonist binding that is uncoupled from adenylate cyclase.Proc Nat! Acad Sci USA 1988;85:5478-82. 44. Strader CD, Sigal IS, Candelore MR, Rands E, Hill WS, Dixon RAE Conserved aspartic acid residues 79 and 113of the beta-adrenergic receptor have different roles in receptor function. J Bioi Chern 1988; 263:10267-71. 45. KhoranaHG. Bacteriorhodopsin, a membrane protein that uses light to translocate protons. J Bioi Chern 1988; 263:7439-42.

Receptor mechanisms. Structure and molecular biology of transmitter receptors.

Details of receptor structure and function that were unavailable as recently as two years ago are now readily obtainable through the application of mo...
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