Article Figures & Data
Figures
- Fig. 1.
Endocytosis pathways involved in GPCR internalization. (A) Clathrin-mediated endocytosis of GPCRs. (1) Ligand-mediated receptor activation leads to conformational changes, for instance, exposing AP-2 binding motifs in the receptor C-tail, and/or post-translational modification of the receptor, such as phosphorylation or ubiquitination. (2a) Most GPCRs are recruited to clathrin-coated pits through arrestins. Binding of arrestin to the phosphorylated receptor C-tail enables interaction of the arrestin C-tail with AP-2 and clathrin. (2b) Additionally, receptors can be recruited to clathrin-coated pits independently of arrestins, through direct interaction with AP-2 or binding of their polyubiquitinated C-tails to epsins. (3) Clathrin is recruited to the plasma membrane by adaptor proteins, such as AP-2 and epsins, to form clathrin carriers upon scission of clathrin-coated pits from the plasma membrane by dynamin. (B) Fast endophilin-mediated endocytosis. (1) Endophilin is concentrated at the plasma membrane through interaction with lamellipodin (Lmpd), which depends on the sequential action of Cdc42, formin-binding protein 17 (FBP17), CIP4, and phosphatidylinositol 3,4,5-trisphosphate 5′ phosphatase (SHIP) 1/2. (2) Interaction of a ligand-activated GPCR with endophilin triggers FEME. (3) FEME carriers are formed upon scission of endophilin stabilized invaginations by dynamin. (C) Endocytosis via caveolae. GPCRs can be recruited to caveolae through direct interaction with caveolin (1a) or via Gαq-mediated interaction with caveolin (1b). (2) Caveolae membrane deformations consist of oligomeric caveolin complexes stabilized by coat-forming cavins and membrane curvature inducing pacsins. Caveolae carrier formation relies on dynamin for membrane scission. For clarity, only a selection of the components involved in the various endocytosis pathways are represented in the schematic. PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PS, phosphatidylserine.
- Fig. 2.
Nonconventional receptor-arrestin interactions that do not lead to arrestin-dependent internalization. (A) Hypothetical interaction between arrestin and the 7TM core of a receptor that stabilizes an intermediate state of arrestin without displacement of the arrestin C-tail from the arrestin N-domain. The clathrin and AP-2 interaction motifs in the arrestin C-tail are still masked, but arrestin blocks G protein activation and could potentially potentiate signaling by scaffolding kinases. This model allows simultaneous interaction of the receptor C-tail with a hypothetical protein (depicted as interactor) that mediates receptor internalization. (B) Catalytic activation of arrestin. Arrestin is activated by a transient interaction with the receptor 7TM core and is stabilized at the membrane in this conformation by binding of PI(4,5)P2. Activated arrestins accumulate in clathrin-coated structures where they can scaffold kinases to enhance signaling.
Tables
- TABLE 1
GPCRs that have been shown to undergo arrestin-independent agonist-induced or constitutive endocytosis
Receptor Mechanism of internalization Cell line Method used to show arrestin independence Reference Arrestin-independent agonist-induced endocytosis β1- and α2a-adrenoceptors and D4 FEME BS-C-1 Indirect evidence (FEME is clathrin- and arrestin-independent) Boucrot et al., 2015 β2-adrenoceptor and AT1A Caveolae (partial arrestin-dependence) HEK293, COS-7 Dominant negative arrestin (arrestin-2-V53D) Guo et al., 2015 5-HT2A Dynamin-dependent HEK293, C6 Dominant negative arrestins (arrestin-2-V53D, arrestin-2319−418, arrestin-3284−409) Bhatnagar et al., 2001; Gray et al., 2001 Apelin Clathrin-dependent HEK293 Dominant negative arrestin (arrestin-2319−418) Pope et al., 2016 BLT1 Dynamin- and GRK2 phosphorylation-dependent RBL-2H3 Dominant negative arrestin (arrestin-2-V53D) Chen et al., 2004b D3 Clathrin- and dynamin-dependent, FEME CHO, HEK293, BS-C-1 Dominant negative arrestins (arrestin-2-V53D, arrestin-3-V54D) Boucrot et al., 2015; Xu et al., 2019 ETA Caveolae (partial arrestin-dependence) HEK293 Arrestin-AP-2 BRET, arrestin-AP-2 complex inhibitor (barbadin), arrestin-2/3 siRNA Okamoto et al., 2000; Hamdan et al., 2007; Beautrait et al., 2017 ETB Unknown (partial arrestin-dependence) HEK293 Arrestin-AP-2 BRET, arrestin-2/3 siRNA Hamdan et al., 2007 FPR1 GRK phosphorylation-dependent MEFs, U937 Arrestin-2/3−/− MEFs, dominant negative arrestin (arrestin-2319−418) Hsu et al., 1997; Gilbert et al., 2001; Vines et al., 2003 GLP-1 Dynamin-dependent and caveolae HEK293, MIN6 HEK293-Δarrestin-2/3, arrestin-3 overexpression Syme et al., 2006; Jones et al., 2018 GLP-2 Caveolae BHK-21, DLD-1 Indirect evidence (dynamin-independent and C-terminal truncated receptor mutant) Estall et al., 2004, 2005 IP Clathrin- and dynamin-dependent HEK293 Dominant negative arrestin (arrestin-2319−418) Smyth et al., 2000 M1 Clathrin- and dynamin-dependent tsA201, MDCK, HEK293 Overexpression of arrestin-2 and -3, dominant negative arrestin (arrestin-2319−418) Lee et al., 1998; Vögler et al., 1998; Shmuel et al., 2007 M2 Phosphorylation- and Arf6 GTPase–dependent tsA201, HeLa Arrestin-2 and -3 overexpression Pals-Rylaarsdam et al., 1997; Delaney et al., 2002 M3 Dynamin-dependent tsA201, HEK293 Overexpression of arrestin-2 and -3, dominant negative arrestin (arrestin-2319−418) Lee et al., 1998; Vögler et al., 1998 M4 Dynamin-dependent, FEME tsA201, BS-C-1 Overexpression of arrestin-2 and -3, dominant negative arrestin (arrestin-2319−418) Lee et al., 1998; Vögler et al., 1998; Boucrot et al., 2015 PAR1 Dependent on clathrin, dynamin, phosphorylation, epsin-1 and AP-2 MEFs, COS-7, HeLa, HEK293 Arrestin-2/3−/− MEFs Paing et al., 2002; Paing et al., 2006; Chen et al., 2011 PAR4 Dependent on clathrin, AP-2 and dynamin HeLa Arrestin-2/3−/− MEFs Smith et al., 2016 PKR2 GRK2 phosphorylation- and clathrin-dependent HEK293 Dominant negative arrestins (arrestin-2-V53D, arrestin-3-V54D), arrestin-2 and -3 siRNA Yin et al., 2014 Secretin PKA-dependent HEK293 Dominant negative arrestin (arrestin-2-V53D) Walker et al., 1999 UT Unknown HEK293, MEFs Dominant negative arrestin (arrestin-3-V54D), arrestin-2/3−/− MEFs Giebing et al., 2005 Constitutive internalization CaS Unknown (partial arrestin-dependence) HEK293 HEK293-Δarrestin-2/3 Mos et al., 2019 CXCR4 Dynamin- and PKC-dependent HEK293 COS-7 Indirect evidence (C-terminal truncated mutant) Signoret et al., 1997, 1998 ADGRA3 Clathrin-dependent HEK293 HEK293-Δarrestin-2/3 Spiess et al., 2019 M3 and β2-adrenoceptor MHC-I HeLa, COS-7 Indirect evidence (clathrin-independent) Scarselli and Donaldson, 2009 mGlu5 Dynamin-dependent, caveolae COS-7 Indirect evidence (clathrin-independent) Fourgeaud et al., 2003 mGlu7 MHC-I HeLa Indirect evidence (clathrin-independent) Lavezzari and Roche, 2007 PAR1 Clathrin- and AP-2-dependent MEFs, COS-7, HeLa, HEK293 Arrestin-2/3−/− MEFs Paing et al., 2002, 2006 TPβ Clathrin- and dynamin-dependent HEK293, CHO Dominant negative arrestin (arrestin-3201−409) Parent et al., 2001 US28 Clathrin-dependent HeLa, MEFs Arrestin-2/3−/− MEFs Fraile-Ramos et al., 2003 XCR1 Unknown HEK293 HEK293-Δarrestin-2/3 Bauer et al., 2019 Arf6, ADP-ribosylation factor 6; BHK-21, baby hamster kidney fibroblasts; MDCK; Madin-Darby canine kidney; PKA, protein kinase A.
