Endocytic Function of PSMA

Prostate-specific membrane antigen, like other cell surface receptors, undergoes inter-nalization constitutively, and such spontaneous internalization is enhanced threefold in a dose-dependent manner by PSMA-specific monoclonal antibody J591 (32). It has been shown very clearly biochemically (by using biotinylated cell surface PSMA followed by internalization of the protein), by immunofluorescence analysis or immunoelecton microscopy, that PSMA or the PSMA-antibody complex undergoes internalization through cla-thrin-coated pits and closely resembles the internalization pathway of transferrin receptor and finally ends up in the lysosomes. Such constitutive internalization of PSMA might reflect the recycling of a structural protein or be mediated by binding of a ligand. A detailed characterization of antibody-mediated PSMA internalization revealed the resemblance with the epidermal growth factor receptor (EGFR) with its ligand (33). It is well known that many ligands and their transmembrane receptors are internalized through clathrin-coated pits (receptor-mediated endocytosis). Formation of antibody-antigen complexes on the cell surface often results in internalization through a pathway closely resembling the receptor-mediated endocytosis of peptide hormones, growth factors, and natural ligands (34) (Fig. 2). It can be speculated from these findings that PSMA might have a transport function for a yet unidentified ligand. A monoclonal antibody such as J591 acts as surrogate ligand-inducing internalization.

Targeting internalization of receptors through coated pits and their traffic through endocytic compartments are mediated through specific signals or motifs located on the cytoplasmic tail of the receptors. There are two major classes of sorting signal that mediate

Psma Internalization

Fig. 2. Spontaneous or antibody-induced internalization of PSMA through clathrin-coated pits. The proteins can either recycle through the recycling endosomal compartment (REC) and go to the plasma membrane or they can go to the lysosomes through late endosomes. The cytoplasmic tail of PSMA contains an internalizaton signal, which enables it to internalize into endosomal vesicles. PM, plasma membrane; TGN, trans-Golgi network; AP, adaptor protein.

Fig. 2. Spontaneous or antibody-induced internalization of PSMA through clathrin-coated pits. The proteins can either recycle through the recycling endosomal compartment (REC) and go to the plasma membrane or they can go to the lysosomes through late endosomes. The cytoplasmic tail of PSMA contains an internalizaton signal, which enables it to internalize into endosomal vesicles. PM, plasma membrane; TGN, trans-Golgi network; AP, adaptor protein.

internalization of membrane proteins from plasma membrane and sort these proteins to endosomes/lysosomes and, finally, to the compartment for peptide loading (35-37). These sorting signals are tyrosine-based NPXY and YXXO motifs (37) and dileucine motif (36). Tyrosine motifs are identified in a variety of receptor molecules like the transferrin receptor, low-density lipoprotein receptor, and asialoglycoprotein receptor (38). Dileucine (or leucine-isoleucine sequence) motif (Fig. 3A and Color Plate 12 following p. 302) is important for internalization and lysosomal targeting was found in the y-5 chain of the T-cell receptor, CD4, IFN-y (39-41). Tyrosine-based motifs interact with adaptor complexes API, AP2, and AP3 (42,43) and dileucine-based motifs bind to the P-subunits of API and AP2; ^-chains of API and AP2 has also been reported to bind to these signals. Apart from this, leucine-based signals of lysosomal protein LIMPII and melanosomal membrane protein tyrosinase have been shown to bind to AP3 (44). PSMA has a dileucine motif present at its cytoplasmic tail. Mutation of first leucine (Leu 4) did not change the internalization of monoclonal antibody (mAb) J591; in contrast, conversion of second leucine (Leu 5) resulted in complete loss of internalization indicating that this leucine is important for the internalization (45; Ghosh et al., unpublished observation, 2003). This implied that the dileucine motif is responsible for the internalization. However the dileucine motif is generally associated with the basolateral targeting of proteins, and PSMA is found at the apical surface of the cell. In the case of PSMA, the first amino acid methionine located five amino acids upstream of the crucial leucine is involved in the internalization, which makes this signal a unique internalization signal "MXXXL" in PSMA. Amino acid residues adjacent to such motifs have been shown to influence its function. The dileucine signal of CD4 is active when adjacent serine residues are phosphorylated (46). The cytoplasmic tail of PSMA has consensus protein kinase C sequence (Thr 14) and has two other hydroxyl-con-taining residues (Thr-8, Ser-10), which might serve as phosphorylation acceptor sites. It remains to be seen how mutation of such a residue affects the internalization function of the protein. A detailed mutational analysis has been carried by Rajasekaran's group, which did not have any effect on the internalization function of PSMA. It is known at least in EGFR (47) that the dileucine motif and its neighboring residues need to form an amphipathic helix with hydrophilic residues pointing toward one surface and hydrophobic residues pointing toward the other for the interaction with adaptor proteins needed for sorting. The predicted protein structure of the PSMA N-terminal cytoplasmic tail showed that this region (residues N3 to R19) has a probability to take up an a-helical structure, and the helical wheel projection of this region showed that this helix is an amphipathic a-helix with hydropho-bic residues projecting toward one surface and hydrophilic residues toward the other (Fig. 3B) (48).

A cytoplasmic leucine-based motif has been shown to be involved in the lysosomal targeting of several membrane proteins (39,47,49,50). PSMA colocalizes with lysosomal marker Lampl in LNCaP cells (endogenously express PSMA) or Cos cells expressing transfected PSMA (45) or PC3 cells ectopically expressing PSMA (Ghosh et al., unpublished observation), indicating that PSMA is being localized within the lysosome. Furthermore, swapping the MWNLL (the first five amino acids of the PSMA sequence) and MWNLA (fifth leucine has been changed to alanine) to Tac antigen, this group has shown that the wild-type motif could transport the Tac antigen (which is not a lysosome resident) to the lysosome whereas the mutant motif could not, indicating that MXXXL signal in PSMA is, indeed, a lysosomal signal. Knowledge ofPSMA's internalization function and regulation can be exploited in cancer therapeutics. A detailed analysis is required to define the putative natural ligand for internalization and what role it plays on the biological function of PSMA. Our lab has shown that substrates and antagonists of the carboxypeptidase function do not alter the rate of internalization, which shows that internalization and enzymatic function are two independent processes. Furthermore, it will be interesting to find out if there is a natural ligand for internalization that could substitute for the mAb in a targeted therapy approach.

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