Title

Biological function of cell surface calreticulin of mouse B16 melanoma cells

Date of Completion

January 1996

Keywords

Biology, Molecular|Biology, Cell|Health Sciences, Oncology

Degree

Ph.D.

Abstract

B16 mouse melanoma cells adhere to and spread on laminin. Cell spreading is uncoupled from adhesion when unglycosylated laminin is used as a substratum, and is specifically restored by oligomannosides. The primary objective of this study was to identify and characterize a cell surface lectin which recognizes laminin oligomannosides and then initiates cell spreading.^ The oligomannoside receptor was isolated by mannan affinity chromatography and its sequence is identical to mouse calreticulin. The melanoma cell surfaces become specifically labeled with anti-calreticulin antibodies; such antibodies also inhibit cell spreading on laminin but cell adhesion is unaffected. Melanoma calreticulin, previously adsorbed to a glycosylated laminin surface, also prevents cell spreading by competing with cell surface calreticulin.^ Two calreticulin isoforms, 52 and 62 kDa, are found in non-adherent cells; the 62 kDa isoform separates into a doublet. The relative abundance of each isoform varies in microvesicles derived from plasma membrane, ER membrane and Golgi membrane. Novel isoforms, 75 and 125 kDa, appear in the focal adhesions of laminin-adherent cells. The 75 kDa isoform also appears in the plasma membrane of cells which attach to surfaces and particles devoid of laminin. Analogues of calreticulin isoforms are present in the sera of various animals.^ Surface calreticulin co-immunoprecipitates from intact non-adherent cells with $\beta$1 integrin. Both receptors cluster together in focal adhesions of laminin-adherent cells. B16 cells rapidly release surface calreticulin into the culture medium and the rate of release is enhanced by GPI-specific lipoprotein lipase. Cell surface calreticulin is also found in various mouse and human carcinoma cells and normal hamster cells.^ These composite results indicate that cell surface calreticulin is a putative mannoside lectin which triggers spreading of laminin-adherent B16 mouse melanoma cells. The data indicate B16 mouse melanoma cells contain multiple calreticulin immunoreactive species, which may account for calreticulin's diverse functions. ^