Cloning and structure-function of the H2 histamine receptor

TiPS -- january 1991/Vol. 121

the transmembrane regions. By contrast, if receptors between subfamilies (e.g. Qrs vs au) are compared, the transmembrane homology drops to -40%. The transmembrane regions appear to constitute the ligand-binding domains and thus ultimately dictate the receptor pharmacology. If this classification approach can be extrapolated to the dopamine receptor family, given their structural and pharmacological similarities, the D2 and D3 receptors are co-members of a distinct dopamine receptor subfamily. In my view, it would make sense to incorporate the nomenclature of the new Da receptor into the existing 4 subcategory. Thus, the first Ds receptor that was clonedra would be referred to as Daa (with Dz~.s and D2a.t. designating the short and long isoforms, respectively) with the Ds receptor being designated 4s. This might be especially helpful since many previous investigations of ‘4 receptom may have actually involved the Ds receptor. Adoption of this nomenclature would also provide a rational basis for the assignment of new dopamine receptors yet to be identified and cloned. Thus, on the basis of their structural and pharmacological profiles, novel dopamine receptor subtypes would either be placed into the existing Dr or Ds subcategories (using A, 8, C, etc. designations) or, if sufficiently divergent, would constitute prototypical membem of new (D3, D,, etc.) dopamine receptor subfamilies. DAVID R. SIBLEY Expcrimvlal ?7wrapc1rrinBrawrk, Nntiotd htifufe of NewoloRicd Disorders 14 Stroke, Bcthrsda,MD 20892. USA.

References 1 Kebrbian, J. W. and Caine, D. B. (1979) Nature 277,93-% 2 V*Lr, L. and Meldolesi, J. (1989) Trrmfs Phamlacof.Sn. 10.74-77 3 Soko)off, P., Martres. M. P. and Schwartz, J. C, (1980) Nn*rytr-SrBnliFIIcbrq’s Arch. Fharmmool. 315,89-102 4 Seeman, I? (1982) Biorhral. Phsrrtmcol. 31.2563-2568 5 Creese, I., Sibley, D. R., Hamblin, M. W. and Leff, 5. E. (1983) Armc. Rrv. NLWOxi. 6, 43-71 6 Monsma. F. 1.. Jr, Mahan, L. C., McVittie, L. D., Gerfen, C. R. and Sibley, D. R. (1990) Pmt. Natf Arad. Sri. USA 87.6723427 7 Dewy, A., Cingrich, J. A., hiardeau, P., Fremwu, R. T.. Jr, Bates, M. D. and Cemn, M. G. (1990) N~frw 347,72-76

9 8 Zhou, Q. Y. rf al. (1990) Natarc .347, 76-80 9 Sunahara, R. K. PI RI. (1990) Naterr 347, 80-83 10 O’Dowd, B. F., Letkowitz, R. J. and Camn, M. G. (1989) Atrnrc. Rrs. Nrlrrosci. 12.67-83 11 Mahan, L. C., Burch, R. M.. Monsma, F. J., Jr and Sibley. D. R. (1990) Pmr. Nat1 Arad. Sri. USA 87.2196-2200 12 FeMer, R. A., Felder, C..C.. Eisner, G. M. and lose, I’. A. (1989) Avr. 1. Physiof.275, F315-f327 13 Bunzow, J. R. cl RI. (1988) Natare 336, 783-787 14 Monsma, F. J., Jr, McVittie, L. D.. Gerfen, C. R., Mahan, L. C. and Sibley. D. R. (1989) Natrrc 342,926-929 15 Gims, B. et a/. (1989) Nnfarr342.923926 16 Grandy, D. K. c1 al. (1989) Pror. Nat/ Acud. Sri. USA 86.9762-9766 17 Selbie, L. A., Hayes, C. and Shine, J.

(1989) DNA 8,683-689 18 Dal Toso. R. CI al. (1989) EM60 /, 8, 4025-1034 19 Chio. C. L.. Hess. C. F., Graham. R. S. and Huff. R. M. (1990) Nnrrrrr 3.13. 266-269 20 O’Malley. K. L.. Mzck. F J . Ch&hn. K. Y. and Todd, ft. D. (1990) 610. Cl?OSiSfry 29, 1367-1371 21 Einhom. L. C., Falardeau. P., Camn, M. C. and Oxford. G. S. (1990) br. Nrtrrosd. Ahsfr. 16, 3B2 22 Mansour, A. cf al. (1990) /. Nrrtrosri. 10, 2587-26GlJ 23 Sokoloff, I’., Gims, 8.. Ma&es, hi. I’.. Bouthenet. M. L. and Schwartz. J. C. (1990) Nntrrrc347, 146-151 Aj76: cir-(+!-(ls,Za)-5 methoxy-l-methylZ-(n-pmpylamino) trtralin lJ= cis-(+)-(ls,2~)-5 methoxy-lmethyl-2-(di-s-pmpylamino) tetralin

Cloning and structur~function of the Ha histamine receptor There are a very limited number of mechanisms by which neurotransmitters and hormones can trigger an intracellular response on binding to their cell surface receptors. Each receptor mechanism and core structure has been well conserved during evolution; however, this is not so for binding site domains and other specific regions where evolutionary variability has ensured large numbers of receptors which belong to a specific gene family but are activated by different ligands. One such superfamily consists of the receptors that catalytically activate heterotrimeric GTPases (G proteins), which themselves

constitute another superfamily of proteins. It now seems likely that there are over 150 different Gprotein coupled receptors, of which -40 have been cloned. Many of these receptors have monoamines as their endogenous ligands e.g. noradrenaline, dopamine, acetylcholine and 5-HT, and have specific conserved residues which are thought to form part of the binding site. These residues are absent in the cloned peptide (e.g. neurokinin) or hormone (e.g. LH) receptors. An important new member of the monoamine receptor family, the H, histamine receptor, has now been cloned’ using a poty-

TiPS -/away

Ill reaction !PCR) merase chain approach’ and cDNA from gastric parietal cell mRNA. The full length clone, obtained by genomic cloning, was used to permanently transfect L-cells. The evidence that the sequence is that of the Hz receptor is based on a numbel or findings: ?? Low

concentrations of histamine (UP-10”~) elevated the CAMP content of these (but not control) cells, the dose-response curve being shifted to the right by the HZ receptor e7tagonist cimetiSine. ?? Histamine-sensitive [3HJtiotidine binding to the transfected cells was detectable and inhibited by cimetidine with the same affinity (l-3 X 10’ M-‘) as found in the cyclase assay. ?? Northern

b!ot analysis showed that the mRNA fur tilis gene product was present in high abundance in canine parietal cells but not detectable in chief cells,

liver, heart, ileum or adrenal glands. Low lcve:s of the mRNA were found in the brain. The H7 receptor sequence is shorter (359 amino acids) than most Cprotein receptor sequences. However it contains the characteristic motifs; seven hydrophobic transmembrane domains (TMs); an Nglycosylation concensus sequence at the amino terminus; the cysteine residues in the first and second outer loops which are considered to form a structurally important disulphide bond’*‘; the acidic residue towards the inside of TM2; and the Asp-Arg-Tyr sequence in the second intracellular loop. In addition, there is an aspartatc residue in TM3 which is found in all monoamine receptors and is thou ht from site-directed 8 mutagenesis. and protein labelling4 studies to be the primary site of binding of the positively charged nitrogen. However there is one important difference between the H, sequence and other catecholamine receptors. Although these sequences show quite extensive homology, the HZ sequence lacks the two serines in TM.5 Mutagenesis studies indicate that these residues form hydrogen bonds with catechol hydroxyl group@ (Fig. la). Instead, there is a threonine residue and an aspartate residue. This is the first time that an

acidic residue has been found in this region. Grant2 et nl.’ postulate that these two residues may be important for hydrogen bonding with the nitrogen atoms of the imidazole ring (Fig. lb). In thi: figure, the assignment of the Hbond donor and acceptor is not clear since the ionization state of the carboxyl residue is not known. it appears that aspartate residues in the hydrophobic transmembrane domains may have high pK values7. Therefore the carboxyl group, if uncharged, and the threonine hydroxyl group, could both act as H-bond donors or acceptors. If the carboxyl group of Asp186 is ionized, it can only act as a H-bond acceptor (B in Fig. 1) and Thrl90 would act as an Hbond donor (A in Fig. 1). There is a fascinating analogy with the postulated aspartateimidazole-threonine interaction with that of the renowned enzyme catalytic triad Asp102-His57Ser195 of chymotrypsin and other serine proteases. In the latter system, protonati;~n/lautomerism at thr imidazole ring is considered to be important for catalysis. In the case of the HZ receptor, tautomerism of the proton on the nitrogen of the imidazole ring of histamine or equivalent residue on other HZ agonists is thought to be an important component in the ability of the agonist to activate the receptor”“.

TiPS Receptor Nomenclature Supplement 1991

With the ability

1992 [Vol. 121 to readily muta-

genize receptors, and the increasing sophistication of the modelling of receptor-ligand interactions, it should be possible, but very challenging, to rationalize the wealth of structure-activity relationships which have been generated in the search for clinically useful Hz antagonists. It will presumably only be a short time before the sequences of HI and Hs receptors and possible variants are available. NIGEL J. hf. BIRDSALL Divisiorr of Php~col Biochmistry, National Irrstitutr for Medical Research, Mill Hilt, LondonNW7 IAA. UK.

References

1 Crank,I. etal. (1990) Pror. Nat\ Acad. Sri. USA (in press) 2 Libert, F. cl al. (1909) Science244,~572 3 Dixon. R. A. F. et a/. (1987) EMBO /. 6, 3269-3275 4 Kurtenbach. E. rt al. (1990) 1. Biol. Chum. 265,13702-13709 5 Strader, C. D. Et al. (lQ88) /. Biol. Chcm. Lb& 10267-10271 6 Strader, C. D., Candelore, M. R., Hill, W. S., Sigal, I. S. and Dixon R. A F. (1989) 1. Biol. Chrm. 264, 33512~13578 7 Birdsall, N. J. M., Ghan. S-C., Eve&h, I’.. Hulme. E. C. and Miller, K. W. (19891 Tmds fhanacol. Sri. 10 (Suppl. Subtypes of Muscarinic Receptms IV), 31-34 8 Durant, C. j., Canellin, C. R. and Parsons, M. E. (1975) I. Med. Chum. 18, 905-w 9 Weinstein, H., Chou, D.. Johnson,C. L., Kang, S. and Green, J. P. (1976) Mol. Pharnracot. 12. 738-745