Mutational analysis of the potential phosphorylation sites in the cytoplasmic domain of integrin beta1A. Requirement for threonines 788-789 in receptor activation.
To investigate the role of the potential phosphorylation sites in the cytoplasmic domain of integrin beta1A, point mutated variants of the protein were stably expressed in the beta1-deficient cell line GD25. Mutants T777A, Y783F, S785A, and Y795F were fully active in promoting cell adhesion, de novo formation of focal contacts, formation of fibronectin fibrils, and activation of focal adhesion kinase. Thus, phosphorylation of these residues is not required for several basic functions of integrin beta1A. On the other hand, the TT788-9AA mutant, was defective in mediating cell attachment and did not contribute to fibronectin fibril formation. The conformation of the extracellular domain was shifted towards an inactive state as measured by binding of the monoclonal antibody 9EG7. Antibody induced clustering of beta1ATT788-9AA demonstrated that the mutant cytoplasmic part was functional in mediating activation of focal adhesion kinase. Therefore, we conclude that threonines 788-789, which are conserved among most integrin beta subunits, are of critical importance for integrin function due to effects on the extracellular conformation of the receptor.
Wennerberg
K
K
Department of Medical and Physiological Chemistry, BMC, Uppsala, Sweden.
Fässler
R
R
Wärmegård
B
B
Johansson
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D003593:Cytoplasm / Q000737:chemistry* / Q000201:enzymology; D004252:DNA Mutational Analysis; D016326:Extracellular Matrix Proteins / Q000494:pharmacology; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D019012:Integrin beta1 / Q000737:chemistry* / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D010641:Phenotype; D010766:Phosphorylation; D011108:Polymers; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011817:Rabbits; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013912:Threonine / Q000737:chemistry* / Q000235:genetics; D016596:Vinculin / Q000032:analysis
D015815:Cell Adhesion Molecules; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D019012:Integrin beta1; D011108:Polymers; D016596:Vinculin; D013912:Threonine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496051:Ptk2 protein, mouse; D011493:Protein Kinase C