The binding of type I collagen to lymphocyte function-associated antigen (LFA) 1 integrin triggers the respiratory burst of human polymorphonuclear neutrophils. Role of calcium signaling and tyrosine phosphorylation of LFA 1.
Monoclonal antibodies to the alpha L beta 2 integrin inhibit the binding of type I collagen to PMN (polymorphonuclear neutrophil leukocytes) as well as the subsequent stimulation of superoxide production and enzyme secretion-elicited by this collagen. Pepsinized collagen still binds PMN but no longer stimulates them. The I domain of the alpha chain of the integrin is involved in the binding. Two sequences of the alpha 1(I) polypeptide chain of collagen participate in the process. Experiments of competitive inhibition by synthetic peptides showed that the sequence RGD (915-917) is used for binding to the cells and DGGRYY (1034-1039) serves to stimulate PMN. Experiments of radioactive labeling of the cells and affinity chromatography on Sepharose-collagen confirmed the presence in PMN extracts of two proteins, 95 and 185 kDa, respectively, corresponding to the molecular weights of the beta 2 and alpha L chains of the integrin and recognized by their specific monoclonal antibodies. The transduction pathways depending on the alpha L beta 2 integrin do not involve a G protein (ruled out by the use of cholera and pertussis toxins), whereas the cytoskeleton was found to participate in the process, as evidenced by inhibition by cytochalasin B. After collagen stimulation, cytoplasmic inositol trisphosphate and calcium ion increased sharply for less than 2 min. The use of the inhibitors staurosporine and calphostin C demonstrated that protein kinase C was involved. Evaluation of the activity of this enzyme showed that, upon stimulation of PMN with collagen I, it was translocated to plasma membrane. Acrylamide gel electrophoresis of the protein bands corresponding to the integrin alpha L beta 2, followed by immunoblotting using monoclonal antibodies to phosphotyrosine, permitted us to demonstrate that, prior to stimulation by type I collagen, there was no phosphorylation, whereas after stimulation, both alpha L and beta 2 chains were stained by anti-phosphotyrosine antibodies. The adhesion of PMN to pepsinized type I collagen triggered tyrosine phosphorylation of the beta 2 chain of the integrin, without stimulating O2-. production by these cells, whereas their stimulation by complete type I collagen induced the tyrosine phosphorylation of both alpha L and beta 2 subunits. The tyrosine phosphorylation of both integrin subunits during transduction of stimuli is a heretofore undescribed phenomenon that may correspond to a new system of transmembrane communication.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015703:Antigens, CD / Q000276:immunology / Q000502:physiology; D018821:CD18 Antigens / Q000276:immunology / Q000502:physiology; D002118:Calcium / Q000097:blood*; D002448:Cell Adhesion / Q000187:drug effects; D003094:Collagen / Q000378:metabolism* / Q000494:pharmacology*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000097:blood; D039423:Integrin alpha1; D007529:Isoflavones / Q000494:pharmacology; D007700:Kinetics; D016169:Lymphocyte Function-Associated Antigen-1 / Q000187:drug effects / Q000502:physiology*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D016897:Respiratory Burst / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction; D013481:Superoxides / Q000097:blood
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018821:CD18 Antigens; D004791:Enzyme Inhibitors; D007074:Immunoglobulin G; D039423:Integrin alpha1; D007529:Isoflavones; D016169:Lymphocyte Function-Associated Antigen-1; D009842:Oligopeptides; D010446:Peptide Fragments; D013481:Superoxides; D019000:Phosphotyrosine; C047981:arginyl-glycyl-aspartic acid; D015544:Inositol 1,4,5-Trisphosphate; D003094:Collagen; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D002118:Calcium