INVESTIGATION OF GROWTH-HORMONE RELEASING HORMONE-RECEPTOR STRUCTURE AND ACTIVITY USING YEAST EXPRESSION TECHNOLOGIES

Growth hormone releasing hormone (GHRH) is the positive regulator of g rowth hormone synthesis and secretion in the anterior pituitary. The p eptide confers by binding to a seven transmembrane domain G protein-co upled receptor. transduction proceeds through subsequent G alpha s sti mulation of adenylyl cyclase. To investigate ligand/receptor and recep tor/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 coup led to the yeast pheromone response pathway. GHRH-dependent signal act ivation in this system required the substitution of yeast G alpha prot ein with proteins containing C-terminal regions of G alpha s. A D60G v ariant (analogous to the little mouse mutation) of the receptor failed to respond to agonist. In parallel studies, GHRH(29) and the N-termin al extracellular region of the receptor were expressed as Gal4 fusion proteins in a 2-hybrid assay. A specific interaction between these pro teins was readily observed. The D60G mutation was engineered into the receptor fusion protein. This protein failed to interact with the liga nd fusion, confirming the specificity of the association between unmod ified proteins. These two yeast expression technologies should prove i nvaluable in additional structure/activity analyses of this ligand/rec eptor pair as well as other peptide ligands and receptors.