Robert H. Oakley, Stephane A. Laporte, Jason A. Holt, Larry S. Barak, and Marc G. Caron

1. The Journal of Biological Chemistry Vol. 274, No. 45, pp. 32248-32257,1999 Association of -Arrestin with G Protein-coupled Receptors during Clathrin-mediated Endocytosis Dictates the Profile of Receptor Resensitization Robert H. Oakley, Stephane A. Laporte, Jason A. Holt, Larry S. Barak, and Marc G. Caron Prepared by DaWoon Cheong

2. Introduction GRK-phosphorylation/ arrestin binding : uncouling, desensitization Clathrin coated pit : pinch off –dynamin - sequestration Agonist binding ? Ref. Seminars in Cell & Developmental biology 9,1998

3. Introduction • Physiological responses - balance between GPCR signal generation and signal termination. - the understand of resensitization :to regulate the physiological or pathological responsiveness of GPCRs • The responsiveness to most GPCR-activating ligands • regained, the biochemical and kinetic properties mediating resensitization • may differ considerably among receptors. • The molecular mechanisms governing the rate at which receptors recycle and re-establish agonist responsiveness are poorly understood. • Regulation of rhodopsin dephosphorylation by arrestin : JBC. Vol. 264 (1989) • A central role for -Arrestins and clathrin-coated vesicle mediated endicytosis in 2AR resensitization : JBC. Vol. 272 (1997) • Cellular trafficking of G-protein coupled receptor/ -Arrestin endocytosis complex : JBC. Vol.274 (1999)

4. Introduction • The dephosphorylation of GRK-phosphorylated receptors - in early endosomes, appears to be a critical step in the resensitization pathway. • An event presumably necessary for the dephosphorylation of GRK-phosphorylated of GPCRs is their dissociation from -Arrestins - Visual arrestin to GRK-phosphorylated rhodopsin prevents rhodopsin dephosphorylation. (JBC. Vol.264, 1989) • Common assumption --Arrestins do not dissocate from desensitized receptors at the plasma membrane but traffiv with them into early endosomes. • But, according to recent observation, receptors specific… • The ability of -Arrestins to remain associated with some receptors but not others suggests that -Arrestins may regulate the cellular trafficking and dephosphorylation of receptor and ultimately their kinetics of resensitization.

5. Introduction • -Arrestins : intracellular protein - Interaction with phosphorylated GPCRs uncouples the receptors from heterotrimeric G proteins, producing a nonsignaling, desensitized receptor. (desensitization ) - Target the GPCRs to clathrin-coated pits for endcytosis to function as docking proteins that link receptors to components of the endocytic machinery such as AP-2 and clathrin (internalization ) - Regulate the dephosphorylation of Receptors (resensitization). • The 2AR and V2R share many of the same signaling properties - agonist-activated receptors coupled to Gs - stimulate adenylyl cyclase - desensitize in a GRK-dependent fashion - internalize into early endosome • But, differ markedly in their ability to recycle and resensitize - 2AR recycles rapidly back to the plasma membrane fully resensitized - V2R, are retained inside the cell and recycle slowly or not at all.

6. 2AR : MGQ………….LCE………TVPSDNIDSQGRNCSTNDSLL 2AR-V2R : MGQ……….LC.ARGRTPPSLGPQDESCTTASSSLAKDTSS V2R : MLM…………… LC.ARGRTPPSLGPQDESCTTASSSLAKDTSS V2R- 2AR : MLM ………….LCE………TVPSDNIDSQGRNCSTNDSLL" Introduction • Using the mutagenesis and chimeric receptors, the ability of -Arrestins to remain associated with desensitized GPCRs and internalize with them into endosomes dictates the properties of GPCR resensitization. - Receptors and Chinera receptor

7. Receptor 2AR 2AR-V2R V2R V2R- 2AR Kd(nM) Kh = 7.7 ± 0.6 Kl = 360 ± 30 Kh = 7.5 ± 1.0 Kl = 400 ± 30 2.4 ± 0.2 2.5 ± 0.3 Bmax (pmol/mg) 1.7 ± 0.1 1.4 ± 0.1 1.5± 0.2 1.1 ± 0.1 EC50 (nM) 0.3 ± 0.01 0.6 ± 0.15 0.3± 0.1 0.4 ± 0.1 Results • Table IBinding, expression, and adenylyl cyclase activation parameters for the wild-type and chimeric receptors • The chimeris receptors were essentially indistinguishable from their wild-type counterparts with respect to their affinity for agonist, level of expression and half-maximal effective concentration(EC50) for adenylyl cyclase activation.

8. Results Fig.1 Sequestration of the 2AR , V2R and 2AR-V2R and V2R- 2AR chimeras. • HEK-293 cells or COS-7 cells • Transfected with each receptor or together with V53D or K44A / arrestin1 or arrestin2 • following a 30 min exposure to agonist (10M Isoproterenol, 100nM AVP) • to measure the loss of cell surface receptors • To evaluate • the arrestin and clathrin dependence of 2AR and V2R sequestration and • the role of endogenous arrestin in 2AR and V2R sequestration • 2AR and V2R both internalize in a arrestin and clathrin-mediated pathway.

9. Results Fig.2 Cellular trafficking of arr2-GFP with the 2AR , V2R and 2AR-V2R and V2R- 2AR chimeras. • HEK-293 cells •  Co-transfected with plasmid containing the cDNA for arre2-GFP and cDNA for each receptor and their chimera • To visualized arre2-GFP fluorescence in cells before and after treatment with agonist (10M Isoproterenol, 100nM AVP) • Absence of agonist, arre2-GFP was evenly distributed throughout the cytoplasm of cells expressing either 2AR or V2R. (0 min) • addition of agonist, promoted the rapid redistribution from the cytosol to the plasma membrane. (2 min) • A more prolonged exposure to agonist produced a striking difference in the trafficking of arre2-GFP • 2AR : remained at the plasma membrane • V2R : within 2 to 3 min, to endocytic vesicle • Chimera – completely reversed

10. Results Fig.3 Colocalization of arr2-GFP with the internalized 2AR , V2R and 2AR-V2R and V2R- 2AR chimeras. • HEK-293 cells •  Co-transfected with arre2-GFP each receptor / their chimera • Pre-labeled with rhodamine-conj. Anti-HA mouse monoclonal ab • treated for 15 min at 37C with agonist (10M Isoproterenol, 100nM AVP) • confocal visualizations of the receptor(red) and the arre2-GFP (green) in same living cells. Colocalization (yellow) fo the receptor with arre2-GFP • V2R : extensive colocalization • 2AR : not colocalization • Chimeras : reversed… • the endocytic pathways of the 2AR and V2R share a common recruiment of arrestin during initial stages but then diverge. arrestin- 2AR complex dissociates at or close memb. And arrestin-V2AR is intenalization into endocytic vesicles.

11. Results Fig.4 Receptor recycling and the redistribution of arr2-GFP following agonist removal. • HEK-293 cells were co-transfected • treated for 30 min at 37C with agonist (10M Isoproterenol, 100nM AVP) • washed to remove agonist, and matained on ice or incubated at 37C for 0, 15, 30, or 60 min • by flow cytometry, cell surface receptors assessed. • visualization of arre2-GFP after agonist removal. (incubated at 37C for 60 min) • arre2-GFP following agonist removal was examed in this recycling paradime. • 2AR , V2R-2AR : in a punctate pattern at the plasma memb. Of cells expressing the recycling • V2R, 2AR-V2R : localized to endicytic vesicles in cells expressing the nonrecycling

12. Results Fig.5 Resensitization of the 2AR , V2R and 2AR-V2R and V2R- 2AR chimeras following agonist removal. • HEK-293 cells were co-transfected • treated for 15 min at 37C with agonist (10M Isoproterenol, 100nM AVP) • washed to remove agonist, and matained on ice (naïve, desensitized) or incubated at 37C for 1hr (resensitization). • cell memb. were prepared and AC activity measured under basal conditions and in the presence of agonist increasing conc. and 10 M forskolin. (naïve – max.) • 2AR : desensitization- decrease in Vmax. fully resensitized • 2AR-V2R : both decrease and right shift, recovery impaired. • V2R, V2R- 2AR : similar results

13. Results Fig.6 Phosphorylation of the agonist-activated the 2AR , V2R and 2AR-V2R and V2R- 2AR chimeras. • HEK-293 cells were co-transfected • treated for 10 min at 37C with or without agonist (10M Isoproterenol, 100nM AVP) • receptors were I.P. and assayed for phosphorylation • 60 min after agonist removal • 2AR : 48  5% reduction in the phosphorylation of the recycling • V2R- 2AR : 67  7% • V2R, 2AR - V2R : little dephosphorylation

14. Results Fig.7 Dephosphorylation of the 2AR , V2R and 2AR-V2R and V2R- 2AR chimeras following agonist removal. • HEK-293 cells were co-transfected • treated for 10 min at 37C with or without agonist (10M Isoproterenol, 100nM AVP) • washed to remove agonist and matained on ice (naïve, desensitized) or incubated at 37C for 1hr (resensitization). • receptors were I.P. and assayed for phosphorylation • 60 min after agonist removal • 2AR : 48  5% reduction in the phosphorylation of the recycling • V2R- 2AR : 67  7% • V2R, 2AR - V2R : little dephosphorylation • The stability of the arrestin interaction with the carboxly-terminal tail of GPCRs dictates the rate receptor dephosphorylation.

15. Results Fig.8 Cellualr trafficking of arr2-GFP with the V2R into endocytic vesicles is mediated by a cluster of three serine residues at the V2R carboxyl terminus.  Upper panel : amino acid composition of C-term. Of the V2R, 2AR and various mutant receptor.  Lowe panel : distribution of arr2-GFP in HEK-293 cells expressing wild-type or mutant receptors following a 30 min teatment at 37C with agonist (1-5:AVP, 6-9:Iso.) • A cluster of three serine residues located in the V2R carboxyl terminus that mediated the trafficking of arrestin with the V2R endosytic vesicles. • The position of serine cluster within the receptor of a stable receptor- arrestin complex that internalizes into endocytic vesicles

16. Results Fig.9 The proximal cluster of three serine residues in the V2R carboxyl terminus is the principal site of V2R phosphorylation. • HEK-293 cells were co-transfected with V2R, V2R-SSSTSS/AAAAAA, V2R-TSS/AAA or V2R-SSS/AAA • treated for 10 min at 37C with 100nM AVP) • receptors were I.P. and assayed for phosphorylation • The proximal cluster of three serine residues, which that internalize into endocytic vesicles, is the principal sithe of V2R phosphoarylation.