Ž . Molecular Brain Research 63 1999 380–383 Short communication Characterization of a cloned Xenopus laeÕis Serotonin 5-HT receptor 1A expressed in the NIH-3T3 cell line Chiara Cappellini a , Paolo Malatesta a , Barbara Costa b , Silvia Marracci a , Irma Nardi a, ) , Claudia Martini b a Laboratori di Biologia Cellulare e dello SÕiluppo, Dipartimento di Fisiologia e Biochimica, UniÕersita di Pisa, Õia Carducci 13, 56010 Ghezzano, Pisa, ` Italy b Dipartimento di Psichiatria, Neurobiologia e Biotecnologie, UniÕersita di Pisa, Õia Bonanno, 6, 56100 Pisa, Italy ` Accepted 27 October 1998 Abstract In a previous work we isolated a Xenopus 5-HT receptor gene and now report the characterization of this receptor. The 1A Ž . HindIII-XbaI fragment of this gene was cloned into the pcDNA I NEO vector and stably transfected into eukaryotic cells NIH-3T3 . To w 3 x determine the specific 5-HT receptor binding, H 8-OH-DPAT was used as radioligand. The selective 5-HT receptor agonist bound 1A 1A only a single class of saturable high-affinity binding sites with pharmacological characteristics similar to those of the mammalian 5-HT 1A receptor. The effects of X5-HT receptor activation on cell growth were also investigated in stably transfected NIH-3T3 cells. The 1A 5-HT agonist 8-OH-DPAT was found to increase DNA synthesis and accelerated cell growth. q 1999 Elsevier Science B.V. All rights 1A reserved. Keywords: 5-HT receptor; Transfected NIH-3T3 cell; Xenopus laeÕis; BrdU incorporation 1A Ž Although multiple types and subtypes of serotonin 5- . HT receptors have been described in the mammalian wx central nervous system 6 , little information is available regarding 5-HT receptors in amphibians. In a previous study, a clone was isolated from a Xeno- pus laeÕis brain cDNA library which showed an overall amino-acid identity of 73% with the human and rat 5-HT 1A receptor. This structural similarity suggested that this clone Ž . named X5-HT encodes the Xenopus homolog of the 1A wx mammalian 5-HT receptor 7 . In situ hybridization stud- 1A ies on Xenopus embryos and adult brain showed that the X5-HT receptor mRNA is expressed in the central ner- 1A Ž . wx vous system CNS and is developmentally regulated 7. In particular, the finding of X5-HT mRNA expression in 1A the early stages of the serotonergic nuclei differentiation ) Corresponding author. Fax: q39-50-878486; E-mail: inardi@dfb.unipi.it suggested that this receptor might mediate a trophic action of serotonin on developing serotonergic cells through an wx autocrine regulatory pathway 7 . In order to confirm that the protein encoded by the X5-HT gene corresponds to the 5-HT receptor sub- 1A 1A type, we expressed the X5-HT receptor in the NIH-3T3 1A cells and determined its pharmacological characteristics. Moreover, we investigated whether the activation of X5- HT stimulates cell proliferation in non-neural cells, simi- 1A larly to what has been found for the human 5-HT 1A w x receptor 12 . The whole coding sequence and part of 5 X and 3 X untranslated sequences of the cloned X5-HT cDNA were 1A ligated into the unique HindIII– XbaI cloning site of the Ž . eukaryotic expression vector pcDNA I NEO Invitrogen and transfected into NIH-3T3 cells. Cells were transfected with the construct pcDNA I NEO-X1 using DOTAP ac- Ž cording to the protocol provided by the supplier Boeh- . 6 ringer Mannheim , using a total of 5 mg of DNAr10 cells. Transfected cells permanently expressing NEO were 0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0169-328X 98 00305-2