ABSTRACT
It was recently reported that HeLa cells have three Arg-Gly-Asp-dependent collagen receptors that do not appear to be in the integrin family of extracellular matrix receptors and bind to either type I or IV collagen or to type I gelatin. It was our goal to determine how these receptors function in HeLa cell-substratum adhesion. We report here that the sequence of events by which the receptors mediate adhesion to collagen or gelatin is: (1) induction of cell attachment by specific collagen receptor-substratum interactions with culture dishes covalently coated with either type I collagen or gelatin - attachment is inhibited by soluble gelatin; (2) stabilization of attachment by exocytotic upregulation of the receptors to the basal plasma membrane, which was demonstrated by analyzing, during cell adhesion, the redistribution of the collagen receptors among the apical plasma membrane exposed to the culture medium, the basal plasma membrane contacting the culture dish, and an intracellular pool of plasma membrane vesicles; (3) the initiation of cell spreading by receptor clustering and cytoskeletal association. Cell spreading is a threshold effect with regard to the surface concentration of gelatin, indicating that collagen receptor clustering is a precondition to the onset of spreading. Observations consistent with this interpretation of the threshold effect are that cells attach but spread more slowly on a substratum that retards receptor clustering, and that collagen receptors, when viewed by immunofluorescence microscopy, form a punctate pattern of fluorescence in the basal plasma membrane during cell spreading. It is also shown that more collagen receptors co-isolate with nondenaturing detergent-stable cytoskeletal preparations after the collagen receptors have been either clustered by antibodies or gelatin in solution, or by a collagen matrix. This indicates that clustering drives the receptors to bind to the cytoskeleton and is a necessary step in the transition from cell attachment to cell spreading.
