Investigation of growth hormone releasing hormone receptor structure and activity using yeast expression technologies.
Growth hormone releasing hormone (GHRH) is the positive regulator of growth hormone synthesis and secretion in the anterior pituitary. The peptide confers activity by binding to a seven transmembrane domain G protein-coupled receptor. Signal transduction proceeds through subsequent G alpha s stimulation of adenylyl cyclase. To investigate ligand/receptor and receptor/G protein associations, the human GHRH receptor was expressed in a modified S. cerevisiae strain which allows for facile measurement of receptor activity by cell prototrophy mediated by a reporter gene coupled to the yeast pheromone response pathway. GHRH-dependent signal activation in this system required the substitution of yeast G alpha protein with proteins containing C-terminal regions of G alpha s. A D60G variant (analogous to the little mouse mutation) of the receptor failed to respond to agonist. In parallel studies, GHRH29 and the N-terminal extracellular region of the receptor were expressed as Gal4 fusion proteins in a 2-hybrid assay. A specific interaction between these proteins was readily observed. The D60G mutation was engineered into the receptor fusion protein. This protein failed to interact with the ligand fusion, confirming the specificity of the association between unmodified proteins. These two yeast expression technologies should prove invaluable in additional structure/activity analyses of this ligand/receptor pair as well as other peptide ligands and receptors.
Kajkowski
E M
EM
American Cyanamid Company, Princeton, New Jersey 08543, USA.
Price
L A
LA
Pausch
M H
MH
Young
K H
KH
Ozenberger
B A
BA
D000818:Animals; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins; D006801:Humans; D051379:Mice; D010675:Pheromones / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D018013:Receptors, Neuropeptide / Q000737:chemistry* / Q000378:metabolism; D018017:Receptors, Pituitary Hormone-Regulating Hormone / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction
D005656:Fungal Proteins; D044385:GTP-Binding Protein alpha Subunits; D010675:Pheromones; D018013:Receptors, Neuropeptide; D018017:Receptors, Pituitary Hormone-Regulating Hormone; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D019204:GTP-Binding Proteins; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs; D020962:Heterotrimeric GTP-Binding Proteins; C048250:somatotropin releasing hormone receptor