Biochemical Society Transactions Z7 Kim, D. (1991) Pfltigers Arch. 418, 338-345 28 Stangl, D., Muff, R., Schmolock, C. and Fisher, J. A. (1993) Endocrinology t32,744-750 29 Beaumont, K., Pittner, R.4., Moore, C. X., Wolfe_ Lopez, D., Prickett, K. S., Young, A. A. and Rink, T. J. (1995) Br. J. Pharmacol. il5, 713-715 30 Wookey, P. J., Tikellis, C., Du, H.-E., ein, H.-F., Sexton, P. M. and Cooper, M. E. (1996) Am. J. Physiol. 27 0, F 289 -F29 4 31 Sexton, P. M., Paxinos, G., Kenney, M. A., Wookey, P. J. and Beaumont, K. (1994) Neuroscience 62, JJJ_JO,/ 32 Sexton, P. M., McKenzie, J. S. and Mendelsohn, F. A. (1988) Neurochem. Int. t?,323-335 33 Deems, R. O., Deacon, R.\ M. and young, A. A. (1991) Biochem. Biophys. Res. Commun.774, 716-720 34 Aiyar, N., Baker, E., Martin, J.,patel,A., Stadel, J. M., Willette, R. N. and Barone, F. C. (1995) J. Neurochem. 65. 1131-1 138 35 Zimmerman, ĮJ., Fisclrcr, J. Ą. arrtl Mtrl'l', I{. (1995) Peptides t6,421-424 36 Zjmmerman, ĮJ., Fischer, J. A., I,'r.ci, I(., ļ "ischer, A. H., Reinscheid, R. I{. arrcl Mtrll', ll, (l9()Õ) I}rain Res. 724, 238-2+ 5 37 owļi, A. A., Smith, D. M., Coppt)cl(, ll. Ą., Morgan, D. G.4., Bhogal, ll., Ghatci, M. A. ,rncl Bloom, S. R. (1995) Endocrinology L36,ZlZ7*2134 38 Coppock, H. A., Owiji, A.4., Illoonr, S. l{ . unel Smith, D. M. (1996) Biochem. J. 318, Ž4 l* r45 39 Kapas, S., Catt, K. J. and Clark, A. J. I.,. (199"5) J. Biol. Chem. 270,25344-25347 40 Eguchi, S., Hirata, Y., Iwasaki, H., Sato, I(., Watanabe, T. Y., Innui, T., Nakajirna, I(,, Sakakibara, S. and Marumo, F. (1994) Endocrinology 135, 245 4 -2458 Received 3 March 7997 with other neuropcptide receptors, with ä-opioid and somatostatin SSTR4 being the most similar in this aspect to galRs. Galanin-galR interactions The N-terminus of galanin (14 amino acids) is absolutely conservcd in all species studied so far. The major pharrnacophores for the central ner- vous System gallĮ are Trpz, Asns and Tyr9. The N-terminal fragment, galanin-(1-16) has only one order of magnitude lower affinity for central neryous Systcm gallĮs than galanin itself. GalR in the gastrointestinal tract probably belongs to another subĻpe, aS has been suggested for the receptor from jcjunal smooth muscle, where the C-terminal part of galanin is necessary for ligand recognition by galR. Galanin-galR interaction has been studied by means of receptor muta- genesis in the Bo\.ves' human melanoma cell line [10]. Despitę much effort put into the develop- ment of non-peptide ligands for galR, no low- molecular* mass galR ligands are known. The design of gallĮ įntagonists has so far followed the 'chimaeric pepticle' scheme (for a review, see t11l). Galanin-G-prote:n interaction The association of galR with G-proteins was first shown by the scnsitivity of galaninergic signal tra sduction to the pertussis toxin-catalysed ADP-ribosylation Į2,131 and to GTP and its analogues Į3]. T'hese findings were later con- firmed by solubilization experiments, where galR co-solubilized with a G-protein a-subunit [14,15]. Table I covers the experimental data available for the coupling of galRs to G-proteins G-Protein-Coupled Receptors for Peptide Hormones in the raĪ pancreatic insulinoma cell line Rin m5F. So far, four subtypes of G-proteins have been found to interact with the galRs in this system: Goa1, G;a1, GgZ and G;a3. Signal trans- duction by subunits PZ, P3 and yZ, y4 has been demonstrated to be involved in the galanin-initi- ated cascade of cellular events. Effector systems used by galRs There is much experimental evidence for the use of different effector systems by galRs. For example, in the case of the inhibition of insulin release by galanin in Rin m5F cells, the following mechanisms have been proposed: closure of CaZ* channels [21], stimulation of ATP-dependent K+ channels [ ZZ] , inhibition of adenylate cyclase llZl and direct inhibition of insulin secretion [23]. Recent high-resolution measurements of cell capacitance in single voltage-clamped B-cells demonstrated no relationship between the sup- pression of exoŅosis and the inhibition of volt- age-dependent Caz* currents nor the inhibition of adenylate cyclase activiŅ [24]. Instead, inhibi- tion of insulin secretion by galanin was suggested to be mediated by activation of the Ca2'r-depen- dent phosphatase, calcineurin. Adenylate cyclase is one of the main effector enzymes in galaninergic signal transduction: inhibition of the stimulated cAMP synthesis caused by galanin has been shown in Bowes' cells, Rin m5F cells llZl, rat cerebral cortex [25], rat hypothalamus, ventral hippocampus and the entorhinal cortex Į26] and in dog co| on | Z7l. Inhibition of adenylate cyclase by galanin can result in the regulation of gene expression, as for the proinsulin gene in the pancreas [28]. Galanin-induced opening of ATP-dependent :į1į:į.'i1:i:i'į. i : Iililtlg.7 Role of the third cytoplasmic loop in signal transduction by galanin receptorš K. Saar* t, A. Valkna* f, U. Soomets* t, K' Rezaei* , M. Zorko* * , M. Zilmer-t and Ü. LangeiŅ * Depar1ment of Neurochemistry and Neurotoxicology, S- l069 l Stockhoļm, Sweden, fDepartment of Bioehcmistry, Tartu University, Jakobi 2, EĖ24OO, Tar1u, Estonia, and tDePartment of Biochemistry, Medical Faculty, Ųniversity of Ljubljana, Yrazov trg2, | O00 Ljub|jana, Sļovenia lntroduction Galanin, a Z9-3} -amino acid-long neuroendo- crine peptide, \ryas discovered in 1983 [l]. It and its receptors are ubiquitously distributed throughout the body: the nervous and endocrine systems, respiratory, gastrointestinal and genito_ urinary tracts. There is a growing body of evidence for a regulatory role for galanin in a whole spectrum of physiological processes. Briefly, galanin has been shown to influence cognition, memory, release of neurotransmitters and hormones (acetylcholine, noradrenaline, glutamate, dopamine, insulin, growth hormone and prolactin), mobiliŲ of the digestive tract, nociception, feeding and sexual behaviour (for a review, see [2]). The last few years have pro- duced experimental evidence for a role for gala- nin in reproduction (for a review, see [3]); for example, it regulates growth hormone and pro- lactin secretion in humans [4]. The hypothalamic neuropeptide Y-galanin-opioid network has been proposed to be the key to nutritional infer- Abbreviations used: galR, galanin receptor; GPCR, G-protein-coupled receptor. Volume 25 GTP cross-linking I l6] Cholera toxin-catalysed ADP-ribosylation I I 6a] in combination with immunoprecipitation with specific antisera* Antisense oligonucleotides against G-protein subunits I l7] Application of antibodies against G-protein a-subunits I I 8l Application of antibodļes against G_protein a-subunits Il9] Application of antibodies against G-protein d-subunits l20l tility [5]. Galaninergic signal transelur;tion has been suggested to be a valuable targert įįlr įļrera- peutic intervention in pain signalling, eognitive and feeding disorders and diabetes. ,l.lre Įriolcrgi- cal effects of galanin appear to be nrccļiatcd Į:y a variety of different effector systems. Įįlucielrrtion of these mechanisms is important, frerrn the point of view of both general signal transeluetion and the great pharmacological potential of glrlnni- nergic signal transduction. Galanin receptors Like all neuropeptide receptors known 60 far, galanin receptors (galRs) belong to the sĮļper- family of G-protein-coupled receptor* (GpeRs). The first galR was cloned from the human mela- noma cell line Bowes ln 1994 [6]. So far it has been cloned from human brain [6a], human small intestine [7], rat brain [ B] and the rat insu- linoma Rinl4B cell line [9]. GalR cloned from human brain and small intestine mucosa are 7007o identical, and Bowes' galR differs in 2 bp at the mRNA level. GalR from rat brain is 9Z?o identical with its human counterpart. There is a 30-3Z7o identiŅ in primary structure compared Method Subtypes of G-proteins coupled to GalR G,aZ and Goa I Ggl , Gg2 and G; a3 G.al B2y2 and G.ul B3yĄ G; a I and G;a2 Gią3 G;a2, G; a3 !r įi aii Subtypes of G-proteins interacting with GalR in Rin m5F cell ļ ine * Cholera toxin catalyses the ADP-ribosylation of the pertussrs toxin-sensitļve G_proteins only in receptor_associated conformation. t997