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PMID: 2104857 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Expression and function of chicken integrin beta 1 subunit and its cytoplasmic domain mutants in mouse NIH 3T3 cells.

The Journal of cell biology ·Vol. 110 ·No. 1 ·1990-01-00 ·Pages 175-84

Hayashi Y, Haimovich B, Reszka A, Boettiger D, Horwitz A

Abstract

Chicken integrin beta 1 cDNA and its site-directed mutants were cloned into a mammalian expression vector and introduced into mouse NIH 3T3 cells. Stable transfectants expressing the chicken beta 1 subunit or its site-directed mutants were identified by immunostaining with antibodies specific for the chicken integrin beta 1 subunit. The chicken beta 1 proteins were expressed predominately in the endoplasmic reticulum of transfectants and to a lesser degree in the plasma membrane. Immunoblots and immunoprecipitations, using anti-chicken integrin antibodies, revealed three different sizes of the chicken subunit (90, 95, and 120 kD) and a mouse 140-kD alpha subunit. Immunoprecipitations of the cell surface receptors showed only two peptides, an 120-kD beta 1 and an 140-kD alpha subunit. Antibodies perturbing mouse and chicken integrin-specific cell adhesions were used to demonstrate that the chimeric receptors functioned in adhesion to both laminin and fibronectin. Immunofluorescent staining with antibodies specific for either the chicken or mouse receptors showed that both the wild type and the chimeric receptors localized in focal contacts. Several mutations in the cytoplasmic domain were synthesized and used in the transfection experiments. In one mutant the tyrosine (Tyr 788) in the consensus sequence for phosphorylation was replaced by a phenylalanine. In another the lysine (Lys 757) at the end of the membrane spanning region was replaced by a leucine. Both of these mutants formed dimers with mouse alpha subunits, participated in adhesion, localized in focal contacts, and displayed biological properties indistinguishable from the wild-type transfection. In contrast, mutants containing deletions greater than 5-15 amino acids nearest the carboxyl end in the cytoplasmic domain neither promoted adhesion nor localized in focal contacts. They did, however, form heterodimers that were expressed on the cell surface.

MeSH Terms
Amino Acid Sequence Animals Blotting, Northern Cell Adhesion Cell Membrane/ultrastructure Cells, Cultured Chickens Chimera Cloning, Molecular Cytoplasm/physiology Cytoskeletal Proteins/analysis DNA/genetics Fluorescent Antibody Technique Integrins/analysis,genetics,physiology Macromolecular Substances Mice Mice, Inbred Strains Molecular Sequence Data Mutation Transfection Vinculin
Chemicals
Cytoskeletal Proteins Integrins Macromolecular Substances Vinculin DNA
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Hayashi Y
Department of Cell and Structural Biology, University of Illinois, Urbana 61801.
Haimovich B
Reszka A
Boettiger D
Horwitz A
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.110.1.175
Published
1990-01-00
Pages
175-84
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2115995
Subset
IM
Grants
NCI NIH HHS · CA-16502 · United States
NIGMS NIH HHS · GM-23244 · United States
External Links
PubMed source DOI: 10.1083/jcb.110.1.175 PMC Full Text View journal details
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