13] Use of Xenopus laevis expression system for structure-function analysis of G-protein-coupled receptor for parathyroid hormone and parathyroid hormone-related protein

Abstract

This chapter discusses methods to examine the effects that structural changes in the parathyroid hormone and parathyroid hormone-related protein (PTH/PTHrP) receptor, such as tail truncation and mutations in intracellular loops, have on the signal transduction through both cyclic AMP-and intracellular Ca 2+ -dependent pathways. The Xenopus oocyte expression system, therefore, provides some evidence as to the location of interaction sites in the receptor for the G proteins that signal through adenylate cyclase and PLC activation. Because of the relative ease of measuring both adenylate cyclase and phospholipase C (PLC) activation in Xenopus oocytes, this System is used to examine the effects of specific mutations on the functional properties of the PTH/PTHrP receptor. The injection of exogenous mRNAs encoding receptors or ion channels in oocytes will often lead to the synthesis of full-length proteins, which may then undergo posttranslational processing. Such proteins are then inserted into the plasma membrane in a functional form and can be screened for using an assay of receptor or ion channel function. The properties of oocytes, together with the tools of receptor mutagenesis and chimeric protein construction, have enabled the dissection of the functional roles of specific regions of receptors, ion channels, and other proteins using this system.