Structure-Function Studies of Detergent Solubilized PTH1R-GαSβγ

Abstract

G-protein coupled receptors (GPCRs) are seven-transmembrane signaling proteins that bind and activate heterotrimeric G-proteins by facilitating the exchange of GDP for GTP. The structural and functional consequences of GPCR-G protein binding have been well studied and include a high-resolution structure of the agonist-GPCR-G-protein ternary complex. However, this work has been performed almost entirely in Family A GPCRs, and have not including the pharmacologically relevant peptide binding Family B GPCRs. The Parathyroid Hormone Receptor Type 1 (PTH1R) is a prototypical Family B member that couples with multiple G-proteins including GαSβγ. The PTH1R is a key regulator of extracellular calcium and phosphate homeostasis, and is important in bone healing and regeneration. Structural studies of PTH1R and its lipid modified cytosolic binding partner GαSβγ have been limited by an inability to obtain high yield purifications of either of these proteins. An additional challenge is to obtain purifications conditions that provide detergent compatibility for PTH1R-GαSβγ complex formation. In this study we have successfully identified conditions for high yield purifications of both PTH1R and GαSβγ. Both proteins are 85 - 95% monodispersed in detergent conditions as measured by Dynamic Light Scattering. Radio-ligand binding experiments demonstrate that the proteins are functional. Furthermore, pull-down experiments show the formation of the PTH1R-GαSβγ complex. This complex is formed primarily through interactions of PTH1R with the GαS subunit, with βγ showing little binding in the presence of agonist. Truncation mutants of the PTH1R were also purified for use in identifying GαSβγ essential binding sites. Taken together, this system provides one of the first models for studying the direct interaction of a Family B GPCR with its cognate G protein. Structural studies using isolated proteins are being pursued for x-ray crystallographic study.