Characterization of pituitary growth hormone and its receptor in the green iguana (Iguana iguana) José Ávila-Mendoza a , Martha Carranza a , Ernesto Pérez-Rueda b , Maricela Luna a , Carlos Arámburo a,⇑ a Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro, Qro. 76230, Mexico b Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Campus Morelos, Cuernavaca, Mor. 62210, Mexico article info Article history: Available online 24 April 2014 Keywords: Growth hormone Growth hormone receptor Green iguana Pituitary Order squamata cDNA cloning abstract Pituitary growth hormone (GH) has been studied in most vertebrate groups; however, only a few studies have been carried out in reptiles. Little is known about pituitary hormones in the order Squamata, to which the green iguana (gi) belongs. In this work, we characterized the hypophysis of Iguana iguana morphologically. The somatotrophs (round cells of 7.6–10 lm containing 250- to 300-nm secretory granules where the giGH is stored) were found, by immunohistochemistry and in situ hybridization, exclusively in the caudal lobe of the pars distalis, whereas the lactotrophs were distributed only in the rostral lobe. A pituitary giGH-like protein was obtained by immuno-affinity chromatography employing a heterologous antibody against chicken GH. giGH showed molecular heterogeneity (22, 44, and 88 kDa by SDS–PAGE/Western blot under non-reducing conditions and at least four charge variants (pIs 6.2, 6.5, 6.9, 7.4) by isoelectric focusing. The pituitary giGH cDNA (1016 bp), amplified by PCR and RACE, encodes a pre-hormone of 218 aa, of which 190 aa correspond to the mature protein and 28 aa to the signal peptide. The giGH receptor cDNA was also partially sequenced. Phylogenetic analyses of the amino acid sequences of giGH and giGHR homologs in vertebrates suggest a parallel evolution and functional relationship between the GH and its receptor. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Growth hormone (GH) is expressed primarily in the pituitary of all vertebrates. It has been associated with stimulation of body growth (by promoting cell differentiation and proliferation), and it has other modulatory effects on metabolism, immune and repro- ductive functions, neuroprotection, and behavior, among others (Ahumada-Solórzano et al., 2012; Alba-Betancourt et al., 2011; Scanes, 1995). GH, together with prolactin (PRL), chorionic somato- tropin hormones (CSH), and somatolactin (SL) constitute a family of hormones that show structural homologies at the level of gene organization, indicating that they share a common ancestral genetic origin which then evolved through subsequent duplication and divergence (Kawauchi and Sower, 2006). Growth hormones from different species are composed of around 190 amino acids and their mature primary structures are highly similar, particularly in the domains corresponding to the four a-helices located at posi- tions 7–34, 75–87, 106–127, and 152–183 (as numbered in porcine GH, Abdel-Meguid et al., 1986). This protein has a molecular weight close to 22 kDa and contains four conserved cysteine residues that form two disulfide bonds (Charrier and Martal, 1988). GH has been described as a family of several molecular iso- forms that are the result of gene duplication, alternative mRNA splicing, and post-translational modifications (Arámburo et al., 1990, 2000; Forsyth and Wallis, 2002). The molecular heterogene- ity of GH contributes to its diversity of functions, which include osmoregulation, control of antifreeze molecule synthesis, and regulation of sexual maturation in fish (Riley et al., 2003); modulation of reproduction, steroidogenesis, and aging in birds (Ahumada- Solórzano et al., 2012); and regulation of cell differentiation, apopto- sis, and postnatal growth in mammals (Forsyth and Wallis, 2002; Isaksson et al., 1982; Kopchick, 2003). Little is known, however, about the specific bioactivities of GH in amphibians and reptiles. Growth hormone exerts its actions through binding to its spe- cific receptor (GHR), which is located in the cell membrane of tar- get tissues, such as liver, brain, bone, and muscle (Kopchick and Andry, 2000). The ligand–receptor interaction triggers a series of intracellular signals that end in a biological action in response to GH stimulation (Herrington and Carter-Su, 2001). GHR family members have an extracellular N-terminal ligand-binding domain involved in receptor–receptor dimerization, a single transmem- brane domain, and an intracellular domain that connects the receptor to downstream effector pathways (Brooks et al., 2008). http://dx.doi.org/10.1016/j.ygcen.2014.04.009 0016-6480/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Fax: +52 (442)238 1005. E-mail address: aramburo@unam.mx (C. Arámburo). General and Comparative Endocrinology 203 (2014) 281–295 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen