Cloning and functional characterization of a testicular TSH receptor cDNA from the African catfish (Clarias gariepinus) H F Vischer and J Bogerd Department of Endocrinology, Faculty of Biology, Hugo R Kruyt building, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands (Requests for offprints should be addressed to J Bogerd; Email: j.bogerd@bio.uu.nl) Abstract A cDNA encoding a putative thyroid-stimulating hormone receptor (cfTSH-R) was cloned from the testis of the African catfish (Clarias gariepinus). The cfTSH-R showed the highest amino acid sequence identity with the TSH-Rs of other fish species. In addition, an insertion of approximately 50 amino acids, specific for the TSH-R subfamily, was also present in the carboxy terminus of the amino-terminal extracellular domain of the cfTSH-R. Next to the testis and thyroid follicles, abundant cfTSH-R expression was detected in cerebellum, brain, ovary, seminal vesicles and pituitary, while weaker expression was found in muscle, stomach, intestine, head-kidney, liver, kidney and heart. HEK-T 293 cells, transiently expressing the cfTSH-R, significantly increased intracellular cAMP levels in response to human TSH. Catfish LH, human choriogonadotropin and human FSH were also able to induce this cfTSH-R-mediated response, although with considerably lower efficiency than human TSH. These results indicated that a functional cfTSH-R had been cloned from the testis of African catfish. Journal of Molecular Endocrinology (2003) 30, 227–238 Introduction In fish, as in other vertebrates, the pituitary-derived thyroid-stimulating hormone (TSH) and gonado- tropins – luteinizing hormone (LH) and follicle- stimulating hormone (FSH) – are essential for differentiation, growth and functional regulation of thyroid follicles and gonads respectively. These glycoprotein hormones act on their target tissues via their respective cell membrane receptors. The receptors for TSH, FSH and LH (TSH-R, FSH-R and LH-R respectively) belong to the superfamily of G protein-coupled receptors, and constitute, together with a restricted number of structurally homologous, invertebrate and vertebrate orphan receptors, the subfamily of leucine-rich repeat- containing G protein-coupled receptors (LGR) (Hsu et al. 2000). LGRs are characterized by the presence of multiple leucine-rich repeat (LRR) motifs in their relatively large N-terminal extra- cellular domains, which have been postulated to adopt a horseshoe-shaped conformation to which their respective (glycoprotein) hormones can bind (Kajava et al. 1995, Bhowmick et al. 1996). In fish, the biological activity of TSH is primarily directed to thyroid follicles, which are dispersely situated in the basibranchial region – within the connective tissue on the surface of the ventral aorta – in contrast to the situation in mammals, where the thyroid follicles are encap- sulated into a gland. Moreover, extra-thyroidal TSH-R expression has been reported for several species (e.g. Kumar et al. 2000, Crisanti et al. 2001). In our attempts to clone the testicular cDNA coding for the catfish LH-R (cfLH-R; Vischer & Bogerd 2003), we unexpectedly isolated a catfish TSH-R (cfTSH-R) cDNA fragment. Here we report the isolation and functional characterization of the full-length cfTSH-R cDNA and demonstrate its extra-thyroidal expression in numerous tissues of the African catfish. 227 Journal of Molecular Endocrinology (2003) 30, 227–238 0952–5041/03/030–227 © 2003 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org