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Localization of Collagenase at the Basal Plasma Membrane of a Human Pancreatic Carcinoma Cell Line¹

Department of Pathology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, New York 11794 [U. M. M., B. L.]; Department of Medicine and Research, Veterans Administration Medical Center, Northport, New York 11786 [J. Z.]; and Department of Biochemistry, University of Kansas Medical Center, Kansas City, Kansas 66103 [K. S., H. N.]

We have recently presented biochemical evidence for collagen and gelatin degrading activities associated with plasma membranes of various human cancer cell lines. In this report we describe the localization of interstitial collagenase at the basal plasma membrane of the human pancreatic cancer cell line RWP-1, using immunofluorescence and ultrastructural immunogold labeling techniques. Collagenase was expressed on the extracellular face of the plasma membrane. Furthermore, the immunogold labeling was concentrated on the long, finger-like microvillous projections typically seen on the basal cell surface, while the short, brush-like projections characteristic of the apical cell surface were unlabeled. When the cytoplasmic face of the membrane was made accessible, the number of reactive sites increased markedly, indicating a high concentration of enzyme at the inner surface of the plasma membrane.

When plasma membrane fractions of RWP-I cells were prepared by differential centrifugation, high salt washes virtually failed to extract collagenase activity from the membrane, while detergent extraction with n-octyl glucoside, a detergent used in the purification of integral membrane proteins, yielded soluble collagenase activity. When detergent extracted membrane fractions were passed over an anticollagenase immunoaffinity column, collagenase was specifically bound, as demonstrated by the TC^A and TC^B degradation of type I collagen by the bound material. Gelatinolytic activity did not bind to the column. Furthermore, immunoprecipitation of ¹²⁵I-labeled detergent extracts of tumor membranes yielded a single M_r 55,000 band consistent with the zymogen form of the connective tissue collagenase. These morphological and biochemical findings suggest that collagenase is a tightly associated component of the basal plasma membrane, where it occupies a strategic location for directional proteolysis during cell migration and invasion.

¹ Supported by Grant AR39189 from the NIH and by the Merit Review Grant from the Veterans Administration.

Received 10/13/89. Accepted 7/16/90.

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