Regulation of GPCR activity, trafficking and localization by GPCR-interacting proteins

Abstract

GPCRs represent the largest family of integral membrane proteins and were first identified as receptor proteins that couple via heterotrimeric G-proteins to regulate a vast variety of effector proteins to modulate cellular function. It is now recognized that GPCRs interact with a myriad of proteins that not only function to attenuate their signalling but also function to couple these receptors to heterotrimeric G-protein-independent signalling pathways. In addition, intracellular and transmembrane proteins associate with GPCRs and regulate their processing in the endoplasmic reticulum, trafficking to the cell surface, compartmentalization to plasma membrane microdomains, endocytosis and trafficking between intracellular membrane compartments. The present review will overview the functional consequence of β-arrestin, receptor activity-modifying proteins (RAMPS), regulators of G-protein signalling (RGS), GPCR-associated sorting proteins (GASPs), Homer, small GTPases, PSD95/Disc Large/Zona Occludens (PDZ), spinophilin, protein phosphatases, calmodulin, optineurin and Src homology 3 (SH3) containing protein interactions with GPCRs.

Figure 1

β-Arrestin-dependent endocytosis and signalling of GPCRs. Agonist activation promotes the GRK2-mediated phosphorylation that promotes the translocation and binding of β-arrestins, which serves to uncouple receptors from heterotrimeric G-proteins. β-Arrestins function as adaptor proteins that interact with both clathrin and β2-adaptin promoting the clathrin coated vesicle-mediated endocytosis of many GPCRs. c-Src is recruited to GPCRs as a consequence of its interaction with β-arrestin and couples the receptor to the MAPK pathway. β-Arrestin interactions with a variety of proteins allows for the coupling of GPCRs to a variety of different signal transduction pathways whose activation may be independent of heterotrimeric G-proteins. β-Arr, β-arrestin; P, phosphate.

Figure 2

RAMP protein regulation of calcitonin receptor family cell surface expression and pharmacology. (A) RAMP proteins facilitate the terminal glycosylation of calcitonin receptors and glycosylation may contribute to the receptor ligand binding site to regulate ligand binding specificity. (B) RAMP proteins contribute to the calcitonin receptor family ligand binding site to regulate ligand specificity. (C) RAMP proteins interact with and allosterically modulate the ligand binding specificity of calcitonin receptor family members. (D) RAMP proteins facilitate the terminal glycosylation of calcitonin receptors to facilitate their processing in the ER and transport to the cell surface. (E) In the absence of appropriate RAMP protein expression calcitonin receptors are retained in the ER and are not transported to the cell surface.

Figure 3

Homer proteins function to regulate the association of protein complexes in the post-synaptic density. The Homer Ena/Vasp homology (EVH) domain as a consequence of the dimerization of their coiled-coiled domains couple mGluR1 and mGluR5 to inositol 1,4,5 triphosphate receptors and NMDA receptors via their interaction with SHANK which interacts with a protein complex involving GKAP and PSD95. The AMPA receptor is coupled to PSD95 via an interaction with TARP. The Homer EVH domain also couples mGluRs to the regulation of the actin cytoskeleton.

Figure 4

Role of Rab GTPases in regulating GPCR trafficking. Rab5 plays a role in the formation, trafficking and fusion of endocytic vesicles with the early endosome. Rab5 interacts with the AT₁R, which regulates Rab5 activity. Rab4 regulates the rapid recycling of vesicles containing GPCRs to the cell surface, whereas Rab11 regulates slow vesicular recycling. Rab7 plays a role in targeting GPCRs to the late endosome and lysosomes. Each of these Rab GTPases exhibits the capcity to interact with GPCR cargo proteins.

Figure 5

Schema depicting proposed mechanism of CRFR1-mediated sensitization of 5-HT₂R signaling. (A) Agonist activation of CRFR1 leads to the β-arrestin and clathrin-dependent endocytosis of CRFR1. (B) CRFR1 is internalized to Rab5-positive endosomes, where it is colocalized with constitutively internalized 5-HT₂R. (C) The PDZ binding motifs of both CRFR1 and 5-HT₂Rs are required for the Rab4-positive rapid recycling endosome-dependent and CRFR1-facilitated recycling of internalized 5-HT₂R to the cell surface via a putative interaction with a PDZ domain containing protein. (D) Due to increased 5-HT₂R cell surface expression mediated by CRFR1 activation 5-HT₂R signalling is enhanced. H, hormone.