Ligand‐specific patterns of PTH1R and arrestin3 internalization and trafficking define a novel form of ligand “bias”

Abstract

The type 1 parathyroid hormone receptor (PTH1R) couples to Gs, Gq/11, and Gi/o family heterotrimeric G proteins and binds arrestins, which mediate receptor desensitization and arrestin‐dependent signaling. It is known that ligand structure can “bias” PTH1R signaling by changing the efficiency with which it couples to these downstream effectors. Here, we characterize the effect of ligand structure on the signaling, internalization, and intracellular trafficking of human PTH1R in a common HEK293 cell background using a panel of four structurally distinct ligands. Two ligands, hPTH(1‐34) and hPTH(1‐31), behave as conventional PTH1R agonists, activating both Gs‐cAMP and Gq/11‐Ca2+ signaling; while [Trp1]‐PTHrP(1‐36) is a Gs‐cAMP selective agonist; and [D‐Trp12 Tyr34]‐bPTH(7‐34) is a Gs‐cAMP selective inverse agonist. All four promote arrestin‐dependent ERK1/2 activation, with [Trp1]‐PTHrP(1‐36) and [D‐Trp12 Tyr34]‐bPTH(7‐34) exhibiting the greatest degree of arrestin‐dependence. To investigate the effect of ligand structure on PTH1R internalization and trafficking, we employed a “bystander BRET” approach in which the BRET pair consisted of a Renilla luciferase‐tagged Ras‐like small GTPase (Rab) marking a specific intracellular membrane compartment (Rab 1, Rab2, Rab5, Rab7, Rab9 or Rab11) and PTH1R‐YFP or arrestin3‐YFP. Three ligands, hPTH(1‐34), hPTH(1‐31), and [Trp1]‐PTHrP(1‐36) promoted PTH1R‐arrestin3 binding and clathrin‐dependent internalization, while [D‐Trp12 Tyr34]‐bPTH(7‐34), which fails to stabilize a PTH1R‐arrestin3 complex, did not promote internalization. In response to hPTH(1‐34) or hPTH(1‐31), PTH1R transited sequentially through early (Rab5) and late (Rab7/9) endosomes, before distributing between the slow recycling (Rab1/2) trans‐Golgi/ER network and rapid recycling (Rab11) endosome pool. Arrestin3 trafficking paralleled the receptor, consistent with the known role of arrestins in promoting slow PTH1R recycling and degradation. In contrast, PTH1R stimulated with [Trp1]‐PTHrP(1‐36) did not accumulate in Rab5 or Rab7/9 positive endosomes, instead transiting rapidly into the Rab11 and Rab2 positive pools. Notably, arrestin3 did not accompany the PTH1R, indicating that rapid dissociation of the PTH1R‐arrestin complex changed the pattern of PTH1R sorting in favor of receptor recycling. These results suggest that ligand structure, in particular the rate of ligand‐receptor‐arrestin complex dissociation, can “bias” not only the effector coupling profile of a G protein‐coupled receptor, but also it subsequent intracellular trafficking and rate of degradation/resensitization.Support or Funding InformationNIH grants R01 DK055524 (LML), GM095497 (LML); the MUSC FLIPRTETRA Core S10 RR027777 (LML); the Research Service of the Ralph H. Johnson VA Medical Center, Charleston, SC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.