Protein-Exchange Dynamics at GPCR Micro-Domains: a Case Study with the Parathyroid Hormone Receptor

Abstract

Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic protein that contains two PDZ domains and assembles macromolecular complexes and regulates localization, trafficking and mobility of a number of membrane transporters and receptors. Interactions among NHERF1 and its target proteins are stabilized by the bimolecular interaction of PDZ and PDZ-binding domains. The PTH receptor (PTHR) contains a PDZ-binding domain that enables direct association with NHERF1 and tethers the PTHR to the actin cytoskeleton. After activation, the PTHR is trafficked to clathrin. The fate of NHERF1 is unknown. We used PTHR as a model to identify the fate of NHERF and to determine the dynamic interactions with the PTHR. Fluorescence Recovery after Photobleaching (FRAP) and confocal fluorescence microscopy were used to measure mobility of PTHR and NHERF1 and the behavior of these proteins at the cell membrane. Fluorescent mCherryNHERF and Green Fluorescent Protein (GFP)-tagged PTHR were coexpressed in rat osteosarcoma cells. We observed a significant reduction in the mobility of PTHR in the presence of NHERF1. Upon stimulation of the receptor with PTH, PTHR internalization was triggered, while NHERF1 remained near the cell membrane. Furthermore, when the PTHR was immunologically immobilized at the cell membrane, mCherryNHERF mobility was high and equivalent to that of NHERF with non-immobilized PTHR. However, PTH interaction with PTHR caused a rapid and greater increase of NHERF1 mobility, even when the receptor was completely immobilized at the membrane. These results support the view that NHERF1 exhibits dynamic interactions with the PTHR, with receptor occupancy followed by rapid dissociation of NHERF1.