A novel cytoplasmic tail MXXXL motif mediates the internalization of prostate-specific membrane antigen

Abstract

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the alpha-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative micro2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.

Figure 1.

Schematic representation of PSMA cytoplasmic tail mutants and Tac-PSMA chimera used in this study. Deletions are shown by horizontal arrows, and insertion of additional alanines is indicated. Amino acids converted to alanine or valine are indicated as A and V, respectively. Internalization (INT) positive (+) or negative (–) for the respective constructs is indicated.

Figure 2.

PSMA internalization in COS-7 cells expressing wild-type PSMA (PSMA_wt) and the cytoplasmic tail deletion mutant (PSMA-Δcd). (A–C) Internalization of PSMA_wt and FITC-transferrin. COS cells transiently transfected with PSMA_wt were simultaneously incubated with mAb J591 (A) and FITC-transferrin (B) for 2 h, washed, fixed in cold methanol, and stained with Texas Red-conjugated anti-mouse antibody. Representative medial optical sections are shown. (C) Merged image. The yellow color indicates the codistribution of FITC-transferrin and internalized PSMA. (D–F) COS cells expressing Dynamin K44A and PSMA_wt cDNA were incubated with mAb J591 for 2 h, washed fixed, and stained with FITC-conjugated anti-mouse antibody to detect PSMA (D) and with polyclonal anti-dynamin antibody and Texas Red-conjugated anti-rabbit antibody to detect cells expressing the dynamin mutant (E). (F) Merged image. Note that in cells expressing DynaminK44A, PSMA was not internalized. (G–I) PSMA-Δcd–expressing cells were incubated with mAb J591 (G) and FITC-transferrin (H) as described above. PSMA-Δcd does not internalize and therefore, does not colocalize with internalized transferring (I). Bar, 5μm.

Figure 3.

Internalization of the cytoplasmic tail di-leucine mutants of PSMA. (A, E, and I) Surface expression of PSMA in COS-7 cells expressing PSMA-L4A/L5A, PSMA-L4A, and PSMA-L5A mutants, respectively. Forty-eight hours after transfection, the cells were fixed in paraformaldehyde under nonpermeabilized conditions and labeled with mAb J591 followed by FITC-conjugated anti-mouse antibody and visualized by epifluorescence microscopy. (B, F, and J) Internalization of PSMA mutants. (C, G, and K) FITC-transferrin uptake. (D, H, and L) Merged images of PSMA and FITC-transferrin. Representative medial optical sections are shown. Yellow color in H indicates the codistribution of FITC-transferrin and internalized PSMA. Bars, 10 μm (A, E, and I) and 5 μm (B, C, D, F, G, H, K, and L).

Figure 4.

Kinetic analysis of internalization of PSMA_wt and PSMA-L4A in COS cells. (A and B) Time course of PSMA_wt (A) and PSMA-L4A (B) internalization. Transiently transfected COS cells were incubated with mAb J591 and FITC-transferrin for the indicated time points, as described under EXPERIMENTAL PROCEDURES and stained with Texas Red-conjugated anti-mouse antibody. Representative medial optical sections are shown. Arrows indicate peripheral vesicles containing PSMA and transferrin. Bar, 5 μm. (C) COS cells expressing PSMA_wt, PSMA-L4A, or PSMA-L4A/L5A were incubated with J591 and FITC-conjugated transferrin for 1 h at 4°C, washed, and incubated at 37°C to allow for uptake. The cells were fixed after 30, 60, and 120 min and incubated with Texas Red-conjugated secondary antibody. Uptake of mAbs J591 and transferrin were visualized and quantitated by confocal microscopy as described under EXPERIMENTAL PROCEDURES. PSMA internalization was normalized to transferring uptake. The bars indicate SE of 30–40 cells analyzed for each condition.

Figure 5.

Internalization of Tac and Tac-PSMA chimera. (A and E) Surface expression of Tac. Forty-eight hours after transfection the cells were fixed in paraformaldehyde under nonpermeabilized condition, labeled with mAb 7G7 followed by FITC-conjugated antimouse antibody, and visualized by epifluorescence microscopy. (B–D, F–H) Internalization of Tac and FITC-transferrin. The cells were incubated with mAb 7G7 and FITC-transferrin for 2 h, washed, fixed in cold methanol, and stained with Texas Red-conjugated anti-mouse antibody. Representative medial optical sections are shown. (B and F) Internalization of Tac antibody. (C and G) Uptake of FITC-transferrin. (D and H) Merged images. Yellow color in H indicates the codistribution of FITC-transferrin and internalized Tac. Bars, 10 μm (A and E) and 5 μm (B, C, D, F, G, and H).

Figure 6.

Internalization of Tac-PSMA chimeras harboring mutations in the di-leucine signal. Surface expression as well as internalization of PSMA was performed as described in figure legend 5. (A, E, and I) Surface expression of Tac in COS-7 cells expressing Tac-MWNAA, Tac-MWNAL, and Tac-MWNLA chimeras, respectively. (B, F, and J) Internalization of Tac chimera mutants. (C, G, and K) Uptake of FITC-transferrin. (D, H, and L) Merged images. Representative medial optical sections are shown. Yellow color in H indicates the codistribution of FITC-transferrin and internalized PSMA. Bars, 10 μm (A, E, and I) and 5 μm (B, C, D, F, G, H, J, K, and L).

Figure 7.

Internalization of Tac-PSMA chimeras Tac-AWNAL and Tac-MAWNAL, and of PSMA-MA(5). (A and D) Internalization of Tac chimera mutants and (G) mAb J591 in transiently transfected COS cells. (B, E, and H) Internalization of FITC-transferrin. (C, F, and I) Merged images. Note the lack of codistribution of Tac-chimera mutants or PSMA-MA(5) and FITC-transferrin. Bar, 5 μm.

Figure 8.

Internalization of PSMA_wt in HeLa cells expressing dominant-negative AP-2 complexes. PSMA_wt cDNA was transiently transfected into HeLa cells expressing a tetracycline-repressible dominant-negative mutant of μ2. mAb J591 internalization was monitored in mutant μ2-induced cells (A) and in noninduced cells (D). (B and E) Internalization of FITC-transferrin. (C and F) Merged images. Bar, 5 μm.

Figure 9.

Schematic model of binding of the PSMA internalization motif to μ2 of the AP-2 complex. (A) The endocytic determinant of PSMA might form two pins (methionine at position 1 [black circle] and leucine at position 5 [black square]) that fit into a complementary pocket of a μ2 (gray) associating with the cytoplasmic tail of PSMA. Loss of the side chains of leucine-5 (B) or methionine-1 (C) of the internalization motif might result in an altered association of the adaptor preventing the internalization of PSMA. Similarly, extension of the length of the internalization motif with an additional alanine (D) might prevent the binding of the adaptor protein to the cytoplasmic tail of PSMA and therefore inhibit internalization of the protein.