Volume 22, Issue 15, Pages 1361-1370 (August 2012) Endocytosis of G Protein-Coupled Receptors Is Regulated by Clathrin Light Chain Phosphorylation  Filipe Ferreira, Matthew Foley, Alex Cooke, Margaret Cunningham, Gemma Smith, Robert Woolley, Graeme Henderson, Eamonn Kelly, Stuart Mundell, Elizabeth Smythe  Current Biology  Volume 22, Issue 15, Pages 1361-1370 (August 2012) DOI: 10.1016/j.cub.2012.05.034 Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 Phosphorylation Mutants of CLCb Associate with CHC (A and B) HEK293T cells were transfected with FLAG-tagged CLCbwt, CLCbSallA, CLCbS204A, or CLCbS204D, and lysates were prepared and subjected to an immunoprecipitation protocol using nonspecific mouse IgG (α-N/S) and either mouse anti-FLAG antibodies (A) or anti-CHC (X22) antibodies (B). The immunoprecipitates were probed for the presence of CHC using CHC5.9 antibodies, FLAG using rabbit anti-FLAG antibodies, or CLCs using anti-CLCa and anti-CLCb antibodies. Lysates represent 10% of the input material. Arrows indicate the positions of CHC (190kD), CLCa (23.4kD), CLCb (23kD), and FLAG-CLCb (26kD). (C) Immunofluorescence images of HEK293T cells transfected with CLCbwt, CLCbSallA, CLCbS204A, or CLCbS204D. CHC was detected with X22 antibody (green) and overexpressed CLCb (WT and mutants) was detected with rabbit anti-FLAG antibody (red). Scale bar represents 5 μm. See also Figures S1 and S2. Current Biology 2012 22, 1361-1370DOI: (10.1016/j.cub.2012.05.034) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 Phosphorylation of CLCb on Ser204 Modulates Endocytosis (A) HEK293T cells were transfected with CLCbwt, CLCbSallA, CLCbS204A, or CLCbS204D and the rate of transferrin endocytosis was measured as described in Experimental Procedures. Results are expressed as the percentage transferrin internalized as a function of time, and each point represents the mean ± SEM of at least three independent experiments (∗p < 0.05). (B) HEK293T cells were transfected with CLCbwt or CLCbSallA, and the rate of transferrin recycling was measured as described in Experimental Procedures. Data are expressed as the mean ± SD of two separate experiments each performed in duplicate. (C) Lysates were prepared from HEK293T cells transfected with FLAG-tagged CLCbwt, CLCbSallA, or CLCbS204A and subjected to immunoprecipitation with anti-FLAG antibodies. The immunoprecipitates were subjected to 2D gel electrophoresis and western blotting with anti-FLAG antibodies. A sample of the immunoprecipitate from lysates expressing CLCbwt was incubated with calf intestinal alkaline phosphatase (CIP) as described in Experimental Procedures. Arrows indicate different phosphorylated forms of CLCb that disappear on treatment with CIP. Asterisk indicates a spot that is absent in CLCbS204A. See also Figure S2. Current Biology 2012 22, 1361-1370DOI: (10.1016/j.cub.2012.05.034) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 Overexpression of CLCbS204A Causes a Delay in CCV Uncoating (A) HEK293T cells, transfected with CLCbwt or CLCbS204A or CLCbS204D were incubated with Tx-Red Tfn for 5 min at 37°C. Surface Tx-Red Tfn was acid stripped and the cells were fixed and CHC was visualized using the mAb X22 (green). Arrows indicate an overlap between X22 and Tx-Red Tfn. Scale bar represents 2 μm. (B) Quantification of the degree of overlap of Tx-Red Tfn and X22 in cells overexpressing CLCbwt or CLCbS204A or CLCbS204D. The degree of colocalization in cells overexpressing CLCbwt has been arbitrarily set to 1. Results are the mean ± SEM (CLCbwt: 73 cells; CLCbS204A: 93 cells; CLCbS204D: 100 cells). Values are significantly different at ∗∗p < 0.01 or ∗p < 0.05. See also Figure S3. Current Biology 2012 22, 1361-1370DOI: (10.1016/j.cub.2012.05.034) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 Ser204 Phosphorylation Selectively Affects GRK2-Dependent GPCR Trafficking (A) GRK2 phosphorylates CLCa and CLCb: Coomassie-stained gels (upper) or autoradiograms (lower) of in vitro kinase reactions using either GST-CLCa or GST-CLCb as substrate and GRK2 as kinase. (B) GRK2 but not CK2 phosphorylates CLCb on Ser204: Coomassie-stained gels (upper) or autoradiograms (lower) of in vitro kinase reactions using either GST-CLCbwt or GST-CLCbS204A as substrate and GRK2 or CK2 as kinase. (C) GRK2 is an in vivo kinase for CLCb: western blot of HEK293T cells treated with GRK2 siRNA or mock-treated (upper panel). Lysates were prepared from control HEK293T cells or those transfected with siRNA against GRK2 and which had also been transfected with either FLAG-tagged CLCbwt or CLCbS204A and subjected to immunoprecipitation with anti-FLAG antibody. The immunoprecipitates were subjected to 2D gel electrophoresis and western blotting with anti-FLAG antibodies. (D) Agonist-induced cell surface loss of MOPr is reduced by overexpression of CLCbS204A. HEK293T cells stably expressing HA-tagged-MOPr were untransfected (control) or transiently transfected with CLCbwt, CLCbS204A, or CLCbS204D. Cells were stimulated with morphine (30 μM) or DAMGO (10 μM) for 0–30 min, and changes in cell surface receptor expression determined by ELISA. Surface receptor present at time 0 was taken as 100%. Values are means ± SEM of four independent experiments. Asterisk indicates values that are significantly different from control value at the same time point, as assessed by two-way ANOVA with Bonferroni posttest, p < 0.05. (E) P2Y1 and P2Y12 cell surface loss show differential requirements for CLCb phosphorylation: 1321N1 cells stably expressing HA-tagged-P2Y12 or P2Y1 purinoceptor were untransfected (control) or transiently transfected with CLCSallA, CLCS204A, or CLCwt. ADP-induced (10 μM; 30 min) loss of surface receptor was assessed by ELISA. Expression of CLCbSallA and CLCbS204A significantly reduced ADP-induced cell surface loss of P2Y12, but only CLCbSallA reduced loss of P2Y1. Surface receptor present at time 0 was taken as 100%. Values are means ± SEM of three to seven independent experiments for each construct. Asterisk indicates values that are significantly different from control value at the same time point, as assessed by two-way ANOVA with Bonferroni posttest, p < 0.05. Current Biology 2012 22, 1361-1370DOI: (10.1016/j.cub.2012.05.034) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 5 siRNA-Mediated Knockdown of CLCa or CLCb in 1321N1 Cells Reduces ADP-Induced Loss of Surface P2Y1 and P2Y12 Purinoceptor Cells were transfected with siRNA targetting CLCa or CLCb, as described in Experimental Procedures. (A) Western blot (top panels) indicated significant knockdown of CLCa and CLCb; in two experiments for each, percent knockdown as compared to cells treated with control siRNA was, for CLCa, 96% and 86% for P2Y1 cells, 83% and 78% for P2Y12 cells, and for CLCb 79% and 91% for P2Y1 cells, 82% and 55% for P2Y12 cells. (B) ADP-induced (10 μM; 30 min) loss of surface HA-tagged-P2Y1 or P2Y12 purinoceptor was assessed by ELISA. Surface receptor present at time 0 was taken as 100%. Values are means ± SEM of three independent experiments for each siRNA. Asterisk indicates values that are significantly different from nonspecific siRNA (control) value at the same time point, as assessed by two-way ANOVA with Bonferroni posttest, p < 0.05. Current Biology 2012 22, 1361-1370DOI: (10.1016/j.cub.2012.05.034) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 6 CLCb Phosphorylation Acts as a Discriminator for Different CCP Populations CLCb phosphorylation is important for endocytosis of a range of housekeeping receptors such as transferrin receptor and signaling cargo such as GPCRs. GPCRs such as P2Y1 and P2Y12 enter cells via different populations of CCPs. Overexpression of CLCbSallA has a dominant-negative effect on endocytosis of several GPCRs. Phosphorylation of Ser204 is most important for those cargoes that are dependent on GRK2 for their endocytosis, e.g., P2Y12 and MOPr. This supports the existence of CCP subpopulations, selective for particular cargo. Housekeeping receptors such as transferrin receptor may enter cells through either population. The portal of entry into cells and the dwell time at a particular location is likely to significantly impact on the signaling output of cargoes such as GPCRs. Current Biology 2012 22, 1361-1370DOI: (10.1016/j.cub.2012.05.034) Copyright © 2012 Elsevier Ltd Terms and Conditions