G protein-coupled receptor sorting to endosomes and lysosomes

Abstract

The heptahelical G protein-coupled receptors (GPCRs) belong to the largest family of cell surface signaling receptors encoded in the human genome. GPCRs signal to diverse extracellular stimuli and control a vast number of physiological responses, making this receptor class the target of nearly half the drugs currently in use. In addition to rapid desensitization, receptor trafficking is crucial for the temporal and spatial control of GPCR signaling. Sorting signals present in the intracytosolic domains of GPCRs regulate trafficking through the endosomal-lysosomal system. GPCR internalization is mediated by serine and threonine phosphorylation and arrestin binding. Short, linear peptide sequences including tyrosine- and dileucine-based motifs, and PDZ ligands that are recognized by distinct endocytic adaptor proteins also mediate internalization and endosomal sorting of GPCRs. We present new data from bioinformatic searches that reveal the presence of these types of sorting signals in the cytoplasmic tails of many known GPCRs. Several recent studies also indicate that the covalent modification of GPCRs with ubiquitin serves as a signal for internalization and lysosomal sorting, expanding the diversity of mechanisms that control trafficking of mammalian GPCRs.

Figure 1

Model of GPCR recruitment to clathrin-coated pits (CCPs). The clathrin adaptor AP-2 is first recruited to the plasma membrane at sites enriched in PIP₂ and facilitates the recruitment of more AP-2, clathrin, and alternate clathrin adaptors. Many activated GPCRs are phosphorylated and bind arrestins, which facilitates recruitment into the nascent CCPs. Other GPCRs harbor short linear peptide sequences or are modified with ubiquitin and recognized by alternate clathrin adaptors. The newly formed CCPs gradually invaginate and pinch off from the plasma membrane through the actions of the GTPase dynamin to form a vesicle. Many other accessory proteins and alternate clathrin adaptors are also present in the nascent and forming CCPs.

Figure 2

G protein–coupled receptor trafficking within the endosomal-lysosomal system. Several studies indicate that distinct mechanisms control trafficking of different mammalian GPCRs. (a) PAR1 displays constitutive and agonist-induced internalization that occur independent of arrestins and is differentially regulated by ubiquitination. Constitutive internalization of PAR1 requires AP-2 and is negatively regulated by ubiquitination. Activated PAR1 is phosphorylated, rapidly internalized, and sorted from endosomes to lysosomes through a SNX1-dependent pathway, which is independent of HRS and TSG101, a component of the ESCRT-I machinery. Whether PAR1 enters MVBs (multivesicular bodies) or sorts directly to lysosomes is not known. (b) Activated CXCR4 is ubiquitinated at the plasma membrane by the E3 ubiquitin ligase AIP4. Ubiquitination functions as an endosomal sorting signal and is not required for CXCR4 internalization. Ubiquitinated CXCR4 is concentrated on HRS-positive microdomains together with AIP4. AIP4 mediates ubiquitination of HRS following CXCR4 activation, which is critical for MVB sorting. CISK phosphorylates and inhibits AIP4 activity and thereby inhibits endosomal sorting of CXCR4. VPS4 also regulates the ubiquitination status of CXCR4 and MVB sorting. (c) Agonist induces rapid phosphorylation and ubiquitination of the β₂AR. Arrestin-3 is ubiquitinated by MDM2 and recruited to activated β₂AR, a process required for β₂AR internalization. Once internalized, β₂AR are dephosphorylated and rapidly recycled back to the plasma membrane through a pathway that involves EBP50/NHERF, NSF, and HRS. Whether deubiquitination of β₂AR is involved in the regulated mode of receptor recycling is not known. After prolonged agonist exposure, activated β₂AR sorts from endosome to lysosomes and are degraded through an ubiquitin-dependent mechanism.

Figure 3

The ubiquitin modification of proteins. Ubiquitin (Ub) is attached to its target protein by the sequential action of E1, E2, and E3 enzymes. The E1 (activating enzyme) first activates ubiquitin in an ATP-dependent reaction by forming a thioester bond at its active-site cysteine with the COOH-terminus of ubiquitin. Ubiquitin is then transferred to the active site cysteine of the E2 (conjugating enzyme). The last step is catalyzed by either an E2 with the help of an E3 (RING-finger) or directly by an E3 (HECT-domain), leading to the transfer of ubiquitin to an epsilon amino group of a lysine residue on the target protein forming an isopeptide bond with the C-terminal glycine of ubiquitin. A single ubiquitin can be attached to proteins (Mono Ub) at a single and/or multiple lysine residues on the target protein. Alternatively multiple ubiquitins can be attached to one another forming poly Ub chains (Poly Ub), typically via lysine 48 or 63 linkages. Ubiquitin is removed from target proteins by the action of deubiquitinating enzymes (DUBs).