Inhibition of melanoma cell binding to type IV collagen by analogs of cell adhesion regulator

Integrin-mediated tumor cell adhesion to type IV collagen is believed to play a role in the invasion of basement membrane proteins and the subsequent metastatic process. The cellular protein CAR (cell adhesion regulator) has been proposed to influence integrin-mediated binding to extracellular matrix proteins, including basement membrane (type IV) collagen. Three analogs of the CAR(138-142) have been tested for activity. The first contains the 138-142 sequence (CAR(138-142), Val-Glu-Ile-Leu-Tyr-NH2), the second contains the 138-142 sequence with a phosphorylated Tyr[pCAR(138-142), Val-Glu-Ile-Leu-Tyr(PO3H2)-NH2], and the third contains the reversed 138-142 sequence (rCAR(138-142), Tyr-Leu-Ile-Glu-Val-NH2). When added extracellularly, none of the analogs had a significant affect on cell adhesion to type IV collagen. Using a novel reversible cell permeabilization method, we found that intracellular incorporation of both CAR(138-142) and PCAR(138-142) resulted in inhibition of cell adhesion in a dose-dependent fashion. The IC50 values were similar to 90 and similar to 10 mu M for CAR(138-142) and PCAR(138-142), respectively. Intracellular incorporation of the rCAR(138-142) peptide had no affect on cell adhesion. Fluorescence microscopy of a fluorescein-labeled CAR(138-142) peptide revealed that the reversible permeabilization procedure resulted in the peptides crossing the cell membrane. Affinity chromatography of melanoma cell lysates with PCAR(138-142) or rCAR(138-142) attached to a solid support of magnetic beads suggested that one protein was bound uniquely by PCAR(138-142) Immunoprecipitation analysis identified vinculin, a protein associated with the actin cytoskeleton, as the protein specifically bound by pCAR(138-142). Immunoprecipitation with pp125(FAK)- or beta 1-integrin-derived mAbs gave negative results. Our study suggests that a possible therapeutic approach for inhibition of melanoma cell adhesion adhesion to extracellular matrix proteins is the use of CAR peptide analogs intracellularly.