RESUMEN
The interaction of GHRH with membrane-bound receptors on somatotroph cells of the anterior pituitary is an important step in the regulation of GH synthesis and secretion. The identification of a G protein-coupled receptor for GHRH has made it possible to investigate the pathway by which GHRH regulates pituitary somatotroph cell function. To initiate an analysis of the mechanisms regulating expression and function of the GHRH receptor, the structure of the gene and its promoter region were analyzed. The coding sequence of the rat GHRH receptor gene is contained within 14 exons spanning approximately 15 kb of genomic DNA. Four transcription start sites are located within 286 bp upstream of the initiation codon. The 5' flanking region of the GHRH receptor gene acts as a functional promoter in rat pituitary tumor GH3 cells, and basal promoter activity is enhanced in GH3 and COS7 cells by cotransfection of an expression construct encoding the pituitary-specific transcription factor Pit-1. The rat GHRH receptor gene is subject to at least 1 alternative RNA processing event that generates 2 receptor isoforms differing by 41 amino acids within the third intracellular loop (IL) of the protein. The short isoform of the GHRH receptor is predominant in pituitary cells. The MtT/S pituitary tumor cell line was found to express the GHRH receptor, and different populations of these cells produce predominantly the long or short isoforms of the receptor messenger RNA, suggesting that the alternative splicing can be regulated. Functional analysis of the two GHRH receptor isoforms demonstrates that both bind GHRH, but only the short isoform signals through a cAMP-mediated pathway. Neither receptor isoform is able to stimulate calcium mobilization from internal stores after GHRH treatment. Our findings indicate that the pituitary-specific transcription factor Pit-1 is involved in the somatotroph-specific expression of the GHRH receptor gene and that functionally distinct receptor proteins are generated by an alternative RNA processing mechanism.
Asunto(s)
Ratas/genética , Receptores de Neuropéptido/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Exones/genética , Regulación de la Expresión Génica/fisiología , Genoma , Masculino , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/fisiología , Ratas Sprague-Dawley , Receptores de Neuropéptido/fisiología , Receptores de Hormona Reguladora de Hormona Hipofisaria/fisiología , Transducción de Señal/fisiología , Transcripción Genética/fisiologíaRESUMEN
We now summarize key issues that we have investigated and highlight additional areas that need to be addressed. We are interested in two basic aspects of the GHRH pathway, those occurring in the brain, involving the synthesis of GHRH, and those occurring in the pituitary, involving signaling by GHRH. We have a long-term interest in the activity and regulation of the hypothalamic neurosecretory cells that synthesize GHRH. With respect to human disease, it is interesting that, despite the primal role played by GHRH in growth-hormone secretion, no mutations in the GHRH gene have yet been identified in association with growth disorders. Focusing on the downstream signaling components of the GHRH pathway, we now know quite a lot about the structure of the GHRH receptor and about some aspects of the signal transduction pathways that mediate the actions of GHRH. With respect to human disease, we have found that in an animal model, the little mouse, a mutation of the GHRH receptor results in growth-hormone deficiency and a dwarf phenotype, and there are ongoing attempts in several laboratories to try to identify similar inactivating mutations in the GHRH receptor in patients with isolated growth-hormone deficiency. Conversely, there is also substantial interest in whether activating mutations in this receptor might be identified in patients with growth-hormone-secreting pituitary tumors. We are also interested in whether there are additional receptors that might mediate some of the extrapituitary actions of GHRH. Finally, a major direction we are taking in the laboratory at the present time is toward understanding the developmental, hormonal, and tissue-specific regulation of the GHRH receptor gene.
Asunto(s)
Expresión Génica , Hormona Liberadora de Hormona del Crecimiento/fisiología , Receptores de Neuropéptido/fisiología , Receptores de Hormona Reguladora de Hormona Hipofisaria/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Animales , Encéfalo/fisiología , Enanismo/genética , Hormona de Crecimiento Humana/deficiencia , Humanos , Sistema Hipotálamo-Hipofisario/fisiología , Ratones , Ratones Mutantes , Ratones Transgénicos , Modelos Biológicos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Receptores de Neuropéptido/biosíntesis , Receptores de Neuropéptido/química , Receptores de Hormona Reguladora de Hormona Hipofisaria/biosíntesis , Receptores de Hormona Reguladora de Hormona Hipofisaria/químicaRESUMEN
The molecular characterization of GHRH and the GHRH receptor provides a framework for understanding the hypothalamic regulation of pituitary somatotroph function. The signaling events discerned from our investigation of GHRH receptor structure and function form the basis of a model for GHRH action, which is shown in Fig. 20. GHRH interaction with its seven transmembrane domain Gs-coupled receptor on the somatotroph (step 1) leads to the release of growth hormone from secretory granules (step 2), which is likely to involve a G protein-mediated interaction with ion channels, and to a stimulation of intracellular cAMP accumulation (step 3) (Mayo, 1992; Lin et al., 1992; Gaylinn et al., 1993). In several cell types tested, elevated cAMP leads to the phosphorylation and activation of the transcription factor CREB by protein kinase A (Gonzalez and Montminy, 1989; Sheng et al., 1991), and one target gene for CREB action is the pituitary-specific transcription factor Pit-1 or GHF-1 (step 4) (Bodner et al., 1988; Ingraham et al., 1988; McCormick et al., 1990). Pit-1 is a prototypic POU domain protein that is required for the appropriate regulation of the growth hormone gene in somatotroph cells, thus providing a pathway by which a GHRH signal can lead to increased growth hormone synthesis in the pituitary (step 5). In addition, Pit-1 is likely to directly regulate the synthesis of the GHRH receptor (step 6), in that the receptor is not expressed in the pituitary of dw/dw mice that lack functional Pit-1 (Lin et al., 1992), and a cotransfected Pit-1 expression construct can activate the GHRH receptor promoter in transiently transfected CV1 cells (Lin et al., 1993). It remains to be determined whether additional direct regulation of the GHRH receptor gene in response to the cAMP signaling pathway occurs (step 7). The inhibitory peptide somatostatin presumably interacts with this same signaling pathway through G protein-mediated suppression of the cAMP pathway (Tallent and Reisine, 1992; Bell and Reisine, 1993). In agreement with the importance of this signaling system for normal growth, a transgene encoding a nonphosphorylatable mutant CREB protein, which blocks the function of the endogenous CREB protein, is able to cause somatotroph hypoplasia and dwarfism in mice when its expression is targeted to pituitary somatotrophs (Struthers et al., 1991). Several steps in the signaling pathway leading to growth hormone secretion are subject to disruption, resulting in growth hormone deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)