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 ␣-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 2 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. INTRODUCTIONProstate-specific membrane antigen (PSMA) was originally identified by the monoclonal antibody (mAb) 7E11-C5 raised against the human prostate cancer cell line LNCaP (Horoszewicz et al., 1987). Subsequently, the PSMA gene was cloned (Israeli et al., 1993) and mapped to chromosome 11q (Rinker-Schaeffer et al., 1995). PSMA is a type II membrane protein with a short cytoplasmic N-terminal region (19 amino acids), a transmembrane domain (24 amino acids), and a large extracellular C-terminal portion (707 amino acids) (Israeli et al., 1993) with several potential N-glycosylation sites. Recently, it has been shown that PSMA is homologous to glutamate carboxypeptidase II (85% at nucleic acid level) isolated from rat brain (Coyle, 1997) and has folate hydrolase activity (Pinto et al., 1996;Halsted et al., 1998), and N-acetylated ␣-linked acidic dipeptidase (NAALDase) activity (Carter et al., 1996. The extracellular domain of PSMA shows homology (26% identity at the amino acid level) to the transferrin receptor I (Israeli et al., 1993) and to a recently cloned transferrin receptor II (Kawabata et al., 1999). The functional significance of homology between PSMA and transferrin receptor is not known.PSMA has been the subject of increasing interest in cancer research due to its potential as a diagnostic and therapeutic target for human prostate cancer (Chang et al., 1999a). PSMA is abundantly expressed in prostate cancer cells. Its expression is further increased in higher-grade cancers, metastatic disease, and hormone-refractory prostate carcinoma (Wright et al., 1996;Silver et al., 1997). In addition, PSMA has become the focus of even more intense interest due to the recent findings that it ...

Folate-linked nanoparticle-mediated suicide gene therapy in human prostate cancer and nasopharyngeal cancer with herpes simplex virus thymidine kinase

“…24 The physiological role of PSMA in prostate cancer remains unknown, but PSMA shows hydrolase enzymatic activity with a folate substrate 23 and is internalized via an endocytic mechanism. 25 If PSMA functions as a receptor mediating the internalization of a putative ligand similar to folic acid, this suggests that the folate-linked nanoparticle binds to PMSA and is then taken up via an endocytic mechanism by LNCaP cells as we reported. 17 In PC-3 cells, our study using RT-PCR confirmed the presence of RFC mRNA, but found no FR or PSMA mRNAs (data not shown).…”

PSMA Ligands for PET Imaging of Prostate Cancer

“…In contrast, small-molecule PSMA ligands bind to the active site in the extracellular domain of PSMA and are internalized and endosomally recycled, leading to enhanced tumor uptake and retention and high image quality (24)(25)(26)(27). The most widely used 68 Ga-labeled PSMA ligands for PET imaging are 68 Ga-PSMA-11 ( 68 Ga-PSMA-HBED-CC) and the theranostic agents 68 Ga-PSMA-617 and 68 Ga-PSMA-I&T (28,29).…”

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“…PSMA has an internalization signal that allows internalization of the protein on the cell surface into an endosomal compartment (13). Previous studies reveal that a type of monoclonal antibody against PSMA is available for imaging diagnosis and therapy of prostate cancer (14,15).…”