Cultured human fibroblasts contain a large pool of precursor beta 1-integrin but lack an intracellular pool of mature subunit.
Previous work has shown the presence of an important intracellular pool of beta 1-integrin subunit in human skin fibroblasts as detected with monoclonal antibody DH12 [De Strooper, B., Van der Schueren, B., Jaspers, M., Saison, M., Spaepen, M., Van Leuven, F., Van den Berghe, H. & Cassiman, J. J. (1989) J. Histochem. Cytochem. 37,299-307]. To analyze this more quantitatively, a radioimmunoassay with radioiodinated monoclonal antibody was developed. The total amount of specific binding sites for monoclonal antibody DH12 on skin fibroblasts was between 0.8-1.5 x 10(6)/cell. After permeabilizing the cells with digitonin, a threefold increase in specific binding was observed, which suggested that about 60% of the total amount of beta 1-subunit was localized intracellularly. From pulse/chase experiments, it was deduced that an important pool of precursor subunit, as defined by its sensitivity to endoglycosidase treatment, existed in fibroblasts. Since in steady-state-labeling conditions, at least three to four times more precursor than mature subunit was immunoprecipitated with monoclonal antibody DH12, we suggested that the intracellular pool of beta 1-integrin subunit is mainly precursor pool. This precursor pool contains a degradation compartment and a maturation compartment. Other investigators have found evidence for a recirculating pool of mature integrin in Chinese hamster ovary cells. Therefore, the presence of a recirculating pool of integrin in human fibroblasts was also considered. The data obtained with mAb DH12 showed that less than 10% of the surface pool of integrin was internalized by endocytosis. Since, however, cross linking of beta 1-integrins with polyclonal antibodies leads to rapid endocytosis of most of the integrin, it remains possible that the quantitatively small effect was actually an artefact induced by the divalent mAb. We conclude that the intracellular pool of beta 1-integrins observed in our previous studies consists of precursor and that in skin fibroblasts no mature beta 1-integrin is available intracellularly for rapid quantitative modulations at the cell surface.
De Strooper
B
B
Centre for Human Genetics, University of Leuven, Belgium.
Van Leuven
F
F
Carmeliet
G
G
Van Den Berghe
H
H
Cassiman
J J
JJ
D000118:Acetylglucosaminidase; D000911:Antibodies, Monoclonal; D001345:Autoradiography; D002478:Cells, Cultured; D004072:Digitonin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000032:analysis*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D010084:Oxidation-Reduction; D011498:Protein Precursors / Q000032:analysis*; D012867:Skin / Q000166:cytology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D016023:Integrins; D011498:Protein Precursors; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D004072:Digitonin