Tyrosine phosphorylation induced by integrin-mediated adhesion of retinal neurons to laminin.
Integrin alpha 6 beta 1 is a laminin receptor involved in adhesion and neurite extension of retinal neurons on laminin. The present study was carried out to understand some of the intracellular mechanisms which allow integrin-mediated neurite extension on laminin in primary neuronal cultures. Both integrin-mediated adhesion to laminin and antibody-induced integrin clustering resulted in the increased tyrosine phosphorylation of a 120 kDa polypeptide which was identified as the focal adhesion kinase. The kinetics of phosphorylation and dephosphorylation of this kinase were dramatically different in neurons plated on laminin, than in neurons in which the receptors were clustered with anti-integrin antibodies. To look at possible interactions of the focal adhesion kinase with integrins, we made use of sucrose velocity gradients, which have allowed the identification of a large complex containing the alpha 6 beta 1 laminin receptor. Analysis of the gradients showed that the focal adhesion kinase was not associated with the integrin receptors under these experimental conditions, while about 26% of the c-Src kinase codistributed with the integrin receptor complex, and showed a molecular size and a distribution similar to that of a 59 kDa phosphoprotein comigrating with the alpha 6 beta 1 receptor. Our results suggest that integrin-induced tyrosine phosphorylation is an early intracellular event during neuronal adhesion, and that the integrin-mediated increase in tyrosine phosphorylation of the focal adhesion kinase is not sufficient per se for the induction of neurite outgrowth. Furthermore, our data indicate that Src kinase may be involved in integrin-mediated neuronal interactions with laminin.
Malosio
M L
ML
Department of Biological and Technological Research (DIBIT), S. Raffaele Scientific Institute, Milano, Italy.
De Curtis
I
I
D000818:Animals; D015153:Blotting, Western; D002448:Cell Adhesion; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D039121:Integrin alpha6beta1; D016023:Integrins / Q000502:physiology*; D007797:Laminin / Q000502:physiology*; D016501:Neurites / Q000502:physiology / Q000648:ultrastructure; D009474:Neurons / Q000201:enzymology / Q000502:physiology*; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D017474:Receptors, Laminin / Q000502:physiology*; D012160:Retina / Q000201:enzymology / Q000502:physiology*; D013395:Sucrose; D014443:Tyrosine / Q000378:metabolism*
D039121:Integrin alpha6beta1; D016023:Integrins; D007797:Laminin; D017474:Receptors, Laminin; D014443:Tyrosine; D013395:Sucrose; D011505:Protein-Tyrosine Kinases