Recycling and Resensitization of the Neurokinin 1 Receptor: INFLUENCE OF AGONIST CONCENTRATION AND Rab GTPASES

Graeme S. Cottrell,Martin Steinhoff,Nigel W. Bunnett,Dirk Roosterman,Fabien Schmidlin

doi:10.1074/jbc.m402479200

Graeme S. Cottrell, Martin Steinhoff + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m402479200

Copy DOI

Abstract

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is thus important to understand the mechanism and function of receptor trafficking. We investigated how the SP concentration affects NK1R trafficking and determined the role of Rab GTPases in trafficking. NK1R trafficking was markedly influenced by the SP concentration. High SP (10 nM) induced translocation of the NK1R and beta-arrestin 1 to perinuclear sorting endosomes containing Rab5a, where NK1R remained for >60 min. Low SP (1 nM) induced translocation of the NK1R to early endosomes located immediately beneath the plasma membrane that also contained Rab5a and beta-arrestin 1, followed by rapid recycling of the NK1R. Overexpression of Rab5a promoted NK1R translocation to perinuclear sorting endosomes, whereas the GTP binding-deficient mutant Rab5aS34N caused retention of the NK1R in superficial early endosomes. NK1R translocated from superficial early endosomes to recycling endosomes containing Rab4a and Rab11a, and Rab11aS25N inhibited NK1R recycling. Rapid NK1R recycling coincided with resensitization of SP-induced Ca2+ mobilization and with the return of surface SP binding sites. Resensitization was minimally affected by inhibition of vacuolar H(+)-ATPase and phosphatases but was markedly suppressed by disruption of Rab4a and Rab11a. Thus, whereas beta-arrestins mediate NK1R endocytosis, Rab5a regulates translocation between early and sorting endosomes, and Rab4a and Rab11a regulate trafficking through recycling endosomes. We have thus identified a new function of Rab5a as a control protein for directing concentration-dependent trafficking of the NK1R into different intracellular compartments and obtained evidence that Rab4a and Rab11a contribute to G-protein-coupled receptor recycling from early endosomes.

Highlights

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is important to understand the mechanism and function of receptor trafficking
The NK1R was at the cell surface, and ␤-arrestin 1-GFP was in the cytosol (Fig. 1A)
There was some translocation to a perinuclear compartment, but the NK1R and ␤-arrestin 1 were prominently colocalized in superficial vesicles

Summary

Introduction

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is important to understand the mechanism and function of receptor trafficking. High SP (10 nM) induced translocation of the NK1R and ␤-arrestin 1 to perinuclear sorting endosomes containing Rab5a, where NK1R remained for >60 min. Low SP (1 nM) induced translocation of the NK1R to early endosomes located immediately beneath the plasma membrane that contained Rab5a and ␤-arrestin 1, followed by rapid recycling of the NK1R. Whereas ␤-arrestins mediate NK1R endocytosis, Rab5a regulates translocation between early and sorting endosomes, and Rab4a and Rab11a regulate trafficking through recycling endosomes. In comparison with our understanding of endocytosis, less is known about the mechanisms of intracellular trafficking of GPCRs. Receptors translocate from coated pits to endocytic vesicles and early endosomes, which serve as a transport node for either rapid recycling from endosomes located immediately beneath the plasma membrane (“short pathway”) or slow recycling from sorting endosomes in a perinuclear location (“long pathway”). Our aims were to (a) examine the effects of graded concentrations of SP on the kinetics and pathway of NK1R recycling and resensitization; (b) determine the contributions of Rab5a, Rab4a, and Rab11a to intracellular trafficking and recycling of the NK1R; and (c) investigate the role of these Rabs in resensitization of the NK1R

Objectives

Results

Conclusion