Efficiency of the clathrin-dependent internalization of PRLr is dependent on PRLr ubiquitination. (A) Internalization of endogenous PRLr in 293T cells that were left untransfected (Mock [squares]) or were transfected with shRNA against clathrin heavy chain (shCLA) or green fluorescent protein (shCON [diamonds]) analyzed by the fluorescence-based assay using anti-PRLr antibody as outlined in Materials and Methods. (B) Internalization of HA-tagged PRLr^WT exogenously expressed in 293T cells that were left untransfected (Mock [squares]) or were transfected with shRNA against heavy-chain clathrin (shCLA) or green fluorescent protein (shCON [diamonds]) analyzed by the fluorescence-based assay using anti-HA antibody as outlined in Materials and Methods. (C) Internalization of endogenous PRLr in 293T cells that were left untransfected (Mock [squares]) or transfected with shRNA against β-TrCP2 (shBTR) or green fluorescent protein (shCON [diamonds]) analyzed as outlined for panel A. (D) Internalization of HA-tagged PRLr^WT exogenously expressed in 293T cells that were left untransfected (Mock [squares]) or transfected with shRNA against β-TrCP2 (shBTR) or green fluorescent protein (shCON [diamonds]) analyzed as outlined for panel B. (E) Internalization of a WT or ubiquitination-deficient S349A mutant of HA-tagged PRLr expressed in 293T cells was analyzed as described for panel B. (F) Internalization of a WT or ubiquitination-deficient S349A mutant of Flag-tagged PRLr stably expressed in MCF10A^Δp53 human mammary epithelial cells was analyzed by reversible biotinylation as outlined in Materials and Methods. Results of immunoblotting (IB) analysis of proteins pulled down with Neutravidin beads by use of anti-Flag M2 antibody are shown. “Input” denotes the samples that did not undergo debiotinylation. IP, immunoprecipitation. (G) PRLr cell surface density was determined using anti-HA antibody and secondary anti-mouse HRP-conjugated antibody binding at 0°C in 293T cells expressing either the WT or the S349A mutant. Data (given in arbitrary units [A.U]) were obtained by subtracting the nonspecific background (nonspecific M1 antibody binding to mock-transfected 293T cells), were normalized per total levels of cellular protein (prot), and are presented as means ± SEM [n = 3]. (H) Rates of internalization of WT or ubiquitination-deficient S349A mutant HA-tagged PRLr expressed in 293T cells pretreated with 100 μM monensin (-m) or vehicle (ethanol) (-e) were analyzed as described for panel B.

Efficient postinternalization sorting of PRLr into the lysosomes requires PRLr ubiquitination. (A) Localization of internalized HA-tagged WT PRLr or PRLr^S349A ubiquitin-deficient mutant. These proteins were internalized for 1 h in the presence of PRL, anti-HA antibody, and FITC-conjugated secondary Fab followed by a 4-h chase procedure. Lysosomes and recycling endosomes were labeled with TRITC-dextran and TRITC-transferrin (TfR), respectively. Dextran completely colocalized with Lamp1 (reference 24 and data not shown). Single optical sections of representative cells obtained by laser confocal fluorescence microscopy are depicted. Bar, 10 μm. (B) pH_v of HA-tagged PRLr-containing endosomes/lysosomes at indicated time points following a postinternalization chase procedure using 293T cells transfected with the indicated HA-PRLr constructs (WT or S349A PRLr) and treated (or not) with the indicated ligands (WT PRL or its antagonist, PRL^Δ1-9,G129R). Following the internalization and chase procedures described in Materials and Methods, the endosomal pH was measured by FRIA and the frequency distribution of the pH_v among the indicated numbers of analyzed vesicles (n) was plotted. Means ± SEM (representing the results obtained with three independent experiments) are also denoted.

Polyubiquitination of PRLr promotes its internalization, postinternalization sorting, and lysosomal degradation. (A) Ubiquitination of Flag-tagged PRLr coexpressed with WT ubiquitin (Ub) or K0 ubiquitin mutant in 293T cells analyzed by denaturing immunoprecipitation using M2 antibody followed by immunoblotting (IB) using anti-HA antibody. Levels of PRLr were also analyzed by immunoblotting with M2 antibody (lower panel). (B) Expression of the K0 ubiquitin mutant slows down the degradation of coexpressed Flag-PRLr as measured by a cycloheximide (CHX) chase procedure followed by immunoblotting using M2 antibody. Levels of β-actin (loading control) were also analyzed. (C) Average results from four experiments similar to that represented by panel B are depicted as percentages of PRLr remaining at the indicated time points of a chase procedure. (D) Interaction of Flag-tagged PRLr with an endogenous α-adaptin complex in 293T cells expressing the indicated ubiquitin constructs was analyzed by coimmunoprecipitation (IP) as described for Fig. 3A. The results obtained with a fraction of α-adaptin bound to PRLr (per mille) are depicted in the bottom panel, Vec, vector; IB, immunoblotting; WCE, whole-cell extract. (E) Internalization of HA-tagged PRLr coexpressed with either WT ubiquitin or K0 mutant in 293T cells was analyzed as described for Fig. 2B. (F) pH_v of HA-tagged PRLr-containing endosomes/lysosomes at 4 h of a chase procedure using 293T cells coexpressing the indicated ubiquitin constructs was analyzed as described for Fig. 4B.

Effect of proteasomal and lysosomal inhibitors on the degradation of endogenous PRLr in human T47D cells. (A) Degradation of endogenous PRLr was analyzed by a cycloheximide (CHX) chase procedure (treatment with CHX [50 μg/ml] and PRL [100 ng/ml]) using T47D cells following 2 h of pretreatment with either the proteasomal inhibitor MG132 (MG [25 μM]) or the lysosomal inhibitor MA (10 mM). Cell lysates were analyzed by immunoblotting with antibodies against PRLr and β-actin (β-act) (loading control). DMSO, dimethyl sulfoxide. (B) Degradation of PRLr following pretreatment with the proteasomal inhibitor lactacystin (LC [10 μM]) or MA (10 mM) or a combination of the two was analyzed as described for panel A.

Ubiquitination of PRLr promotes its interaction with components of AP2, which are required for PRLr internalization. (A) Interactions between Flag-tagged PRLr (WT or S349A mutant) and the endogenous α subunit of the AP2 complex in 293T cells (transfected with the indicated plasmids and treated with PRL [50 ng/ml for 5 min]) were analyzed by immunoprecipitation (IP) using M2 anti-Flag antibody followed by immunoblotting (IB) using the indicated antibodies. Material used for immunoprecipitation was normalized to yield comparable levels of PRLr in all lanes. Levels of the α subunit in whole-cell extracts (WCE) were also assessed. (B) Internalization of endogenous PRLr in 293T cells that were left untransfected (Mock [squares]) or were transfected with small interfering RNA against the α-adaptin subunit of AP2 (shAP2) or luciferase (siCON [diamonds]) was analyzed by the fluorescence-based assay using anti-PRLr antibody as outlined for Fig. 2A.

MS analysis of the nature of ubiquitin chain linkages for PRLr. (A) 293T cells were transfected with Flag-tagged PRLr (P) or pcDNA3 empty vector (V). PRLr was immunoprecipitated (IP) using Flag M2 antibody as described in Materials and Methods. Results represent analysis of 1%, 4%, and 70% of Flag-immunoprecipitated material obtained by immunoblotting (WB) with antiubiquitin antibody, silver staining, and colloidal Coomassie staining, respectively. The position of unmodified PRLr is indicated by an arrow. The region corresponding to ubiquitin-PRLr (Ub∼PRLr) that was used for MS analysis is demarcated. (B) The fraction used to demonstrate the presence of the ubiquitin moiety in the analyzed material was examined by immunoblotting using anti-Flag antibody to detect expression of Flag-PRLr. (C) Representative quantification of K48 linkage based on liquid chromatography-SRM. A spectrum of SRM for the PRLr sample, the internal standard GG peptide, was labeled with a heavy stable isotope, and the results are shown as two peaks at 621.6 and 622.6 m/z (dashed lines). The native peptide corresponding to the K48 linkage was displayed as two additional peaks at 617.9 and 618.9 m/z (solid lines). (D) To quantify the native and standard peptide levels, their elution profiles (intensity versus retention time) were generated for a comparison. The peak area reflected the greatest abundance of the peptides. An example of the results of analysis of K48 linkages is depicted. (E) Results of quantification of types and quantities of ubiquitin chains (given in pmoles) appended to PRLr by the MS-based AQUA method as described in Materials and Methods are presented.

K63 polyubiquitin linkages are required for efficient internalization, sorting, and degradation of PRLr. (A) Overall ubiquitination of Flag-tagged PRLr coexpressed with the indicated ubiquitin (Ub) constructs in 293T cells was analyzed by denaturing immunoprecipitation (IP) using M2 antibody followed by immunoblotting (IB) using anti-HA antibody (which recognizes HA-tagged ubiquitin). Amounts of material subjected to immunoprecipitation were normalized in order to load comparable levels of PRLr in all lanes (lower panel). VEC, vector. (B) Degradation of Flag-tagged PRLr coexpressed with the indicated direct ubiquitin mutants in 293T cells was analyzed as described for Fig. 6B. CHX, cycloheximide; β-Act, β-actin. (C) Quantitative analysis of three independent experiments similar to that represented by Fig. 7B. The results of the analysis are presented as percentages of PRLr remaining at indicated time points of a chase procedure. (D) Degradation of Flag-tagged PRLr coexpressed with the indicated reverse ubiquitin mutants in 293T cells was analyzed as in Fig. 6B. (E) Quantitative analysis of four independent experiments similar to that represented by Fig. 7D are presented as percentages of PRLr remaining at indicated time points of a chase procedure. (F) Internalization of HA-PRLr coexpressed with the indicated direct (upper panel) and reverse (lower panel) ubiquitin mutants was measured as described for Fig. 2B. WT ubiquitin, squares; K48R, open triangles; R63K, open circles. (G) pH_v of HA-tagged PRLr-containing endosomes/lysosomes at 4 h of chase in 293T cells coexpressing the indicated ubiquitin constructs was analyzed as described for Fig. 4B.