RESUMO
We have examined the mechanism of signaling by the 67 kDa YIGSR binding protein of laminin and its properties in neuroblastoma cells. Ligand displacement analysis showed that the interaction with the C(YIGSR)3-NH2 peptide amide is of intermediate affinity (1.5 x 10[-7] M). Cross-linking experiments with sulfo-MBS detected an additional protein with a molecular mass of 116 kDa that binds the YIGSR sequence. Incubation of neuroblastoma cells with C(YIGSR)3-NH2 peptide amide or antibody directed against the 67 kDa laminin binding protein induces tyrosine phosphorylation of proteins with a molecular mass ranging from 115 to 130 kDa and another heterogeneous protein group of 32 kDa.
Assuntos
Laminina/metabolismo , Precursores de Proteínas , Receptores de Laminina/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Anticorpos/farmacologia , Sítios de Ligação , Reagentes de Ligações Cruzadas , Humanos , Camundongos , Dados de Sequência Molecular , Neuroblastoma/metabolismo , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol Diacilglicerol-Liase , Fosforilação/efeitos dos fármacos , Proteínas/química , Proteínas/metabolismo , Ratos , Receptores de Laminina/efeitos dos fármacos , Receptores de Laminina/imunologia , Transdução de Sinais , Células Tumorais Cultivadas , Fosfolipases Tipo C/farmacologiaRESUMO
Differentiated human neuroblastoma LA-N1 cells that were exposed to dibutyryl adenosine 3',5'-cyclic monophosphate for 5 days (primed cells) showed increased adhesion to laminin-, fibronectin-, and collagen type I-coated plates as compared to unprimed cells. Moreover, primed cells seemed to adhere best to laminin. The binding site in laminin, mediating cell attachment, was identified as containing the YIGSR sequence, a known cell binding motif, located in the short arm of the B1 chain of laminin. The synthetic peptide amide, C(YIGSR)3-NH2, containing a repeat of this binding motif, inhibited the attachment of neuroblastoma cells to laminin in a competitive manner, and its inhibitory activity was inversely dependent on laminin concentrations. Affinity chromatography of membrane-extracted proteins over an Affi-Gel 10 column conjugated to C(YIGSR)3-NH2, revealed a major YIGSR-binding protein with an apparent molecular mass of 67 kDa. The 67-kDa surface membrane protein was specifically eluted from the column with the soluble C(YIGSR)3-NH2 peptide, but not with an unrelated peptide. Furthermore, no 67-kDa laminin-binding protein was recovered from an unrelated peptide matrix with the free C(YIGSR)3-NH2 peptide. Ligand blot overlay assays with biotin-labeled C(YIGSR)3-NH2 peptide demonstrated that the 67-kDa receptor is indeed a YIGSR-binding protein. This 67-kDa laminin-binding protein appeared to be down-regulated upon differentiation of LA-N1 cells, as indicated by the level of this protein and its mRNA.