RESUMO
Laminin is a potent promoter of neurite outgrowth, and a synthetic peptide of 19 amino acids, PA22-2, from the A chain has been found to promote process formation. Using peptide affinity chromatography, we have identified a 110-kDa, cell surface ligand from both neural cells and brain which binds this sequence. This binding protein does not share immunological identity with the B1 chain of integrin, and reduction does not alter its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody to the 110-kDa protein stained cellular processes in vivo. Sequence analysis of the first 18 amino acids from the amino terminus yielded almost exact sequence identity with nucleolin, a major 110-kDa nucleolar phosphoprotein. Antibody to nucleolin, however, does not interact with the neural-derived, laminin-peptide-binding 110-kDa protein. The 110-kDa protein appears to be a ligand for a specific site on laminin.
Assuntos
Laminina/química , Fatores de Crescimento Neural , Neuritos/fisiologia , Peptídeos/química , Receptores Imunológicos/química , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Membrana Basal/química , Química Encefálica , Camundongos , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Receptores de Laminina , Células Tumorais CultivadasRESUMO
The present study is a detailed kinetic analysis of the synthesis, release and multimerization of fibronectin (FN) in normal and tumor promoter-treated human lung fibroblasts. Pulse/chase and surface labeling experiments were performed to follow the fate of both newly synthesized and preexisting cell-surface FN over time. The majority of FN (80%) left the intracellular compartment within one hour of synthesis. However, the rate of direct secretion was very low and after one hour, 70% of newly synthesized FN was still at the cell surface. This material was primarily dimeric. Dimeric and multimeric (very high molecular weight) FN was detectable at the cell surface and in the medium 4 hours after synthesis. Pulse-labeled FN multimer levels peaked at 12 hours and declined thereafter. After 24 hours, 85% of pulse-labeled FN had been shed into the medium and the labeled FN remaining at the cell surface was primarily multimeric. Surface labeling experiments confirmed that the majority of FN resides at the cell surface prior to release into the medium. One hour treatment with the phorbol ester tumor promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA), stimulated a nine-fold increase in release of preexisting, dimeric cell-surface FN (125I-labeled). The major effect of longer term TPA treatment up to nine hours was continued depletion of dimeric cell-surface FN. Increased release of cell-surface multimeric FN was also stimulated by TPA, but to a much lesser extent. Release of newly synthesized (pulse-labeled) dimeric FN was also stimulated by TPA though much less than pre-existing FN, and TPA treatment produced a small decrease in the steady-state level of multimeric FN. Thus, preexisting cell-surface FN and newly synthesized FN differ dramatically in their susceptibility to TPA treatment.
Assuntos
Fibroblastos/metabolismo , Fibronectinas/biossíntese , Células Cultivadas , Densitometria , Fibronectinas/química , Humanos , Cinética , Pulmão/metabolismo , Peso Molecular , Acetato de TetradecanoilforbolRESUMO
The process of neurite extension is complex and mechanisms involved probably vary depending on the local microenvironment (tissue site, extracellular matrix, neighboring cells, humoral factors, and developmental stage) of the developing or repairing neuron. Laminin contains at least one and perhaps more sites capable of stimulating process formation in a variety of neuronal cell types. This site contains a lysine and is likely located near a complex sugar residue. A complex of laminin and heparan sulfate proteoglycan appears to form a unique site with comparable activity, which is defined by monoclonal antibody recognition. Cell receptor complexes capable of recognizing the fibronectin cell attachment peptide RGD also bind laminin, while antisera to these complexes inhibit neurite outgrowth in some instances. The identification of laminin active sites and corresponding cell receptors could open new approaches to improving nerve regeneration in both the peripheral and central nervous system.