Tomaselli K J, Damsky C H, Reichardt L F
Department of Physiology, University of California, San Francisco 94143-0724.
J Cell Biol. 1987 Nov;105(5):2347-58. doi: 10.1083/jcb.105.5.2347.
Neuronal responses to extracellular matrix (ECM) constituents are likely to play an important role in nervous system development and regeneration. We have studied the interactions of a neuron-like rat pheochromocytoma cell line, PC12, with ECM protein-coated substrates. Using a quantitative cell attachment assay, PC12 cells were shown to adhere readily to laminin (LN) or collagen IV (Col IV) but poorly to fibronectin (FN). The specificity of attachment to these ECM proteins was demonstrated using ligand-specific antibodies and synthetic peptides. To identify PC12 cell surface proteins that mediate interactions with LN, Col IV, and FN, two different antisera to putative ECM receptors purified from mammalian cells were tested for their effects on PC12 cell adhesion and neuritic process outgrowth. Antibodies to a 140-kD FN receptor heterodimer purified from Chinese hamster ovarian cells (anti-FNR; Brown, P. J., and R. L. Juliano, 1986, J. Cell Biol., 103:1595-1603) inhibited attachment to LN and FN but not to Col IV. Antibodies to an ECM receptor preparation purified from baby hamster kidney fibroblastic cells (anti-ECMR; Knudsen, K. A., P. E. Rao, C. H. Damsky, and C. A. Buck, 1981, Proc. Natl. Acad. Sci. USA., 78:6071-6075) inhibited attachment to LN, FN, and Col IV, but did not prevent attachment to other adhesive substrates. In addition to its effects on adhesion, the anti-ECMR serum inhibited both PC12 cell and sympathetic neuronal process outgrowth on LN substrates. Immunoprecipitation of surface-iodinated or [3H]glucosamine-labeled PC12 cells with either the anti-FNR or anti-ECMR serum identified three prominent cell surface glycoproteins of 120, 140, and 180 kD under nonreducing conditions. The 120-kD glycoprotein, which could be labeled with 32P-orthophosphate and appeared to be noncovalently associated with the 140- and 180-kD proteins, cross reacted with antibodies to the beta-subunit (band 3) of the avian integrin complex, itself a receptor or receptors for the ECM constituents LN, FN, and some collagens.
神经元对细胞外基质（ECM）成分的反应可能在神经系统发育和再生中发挥重要作用。我们研究了一种神经元样大鼠嗜铬细胞瘤细胞系PC12与ECM蛋白包被底物之间的相互作用。通过定量细胞黏附试验，发现PC12细胞很容易黏附于层粘连蛋白（LN）或IV型胶原（Col IV），但对纤连蛋白（FN）的黏附较差。使用配体特异性抗体和合成肽证明了对这些ECM蛋白黏附的特异性。为了鉴定介导与LN、Col IV和FN相互作用的PC12细胞表面蛋白，测试了两种针对从哺乳动物细胞中纯化的假定ECM受体的不同抗血清对PC12细胞黏附和神经突生长的影响。针对从中国仓鼠卵巢细胞中纯化的140-kD FN受体异二聚体的抗体（抗FNR；Brown, P. J., and R. L. Juliano, 1986, J. Cell Biol., 103:1595 - 1603）抑制了对LN和FN的黏附，但不抑制对Col IV的黏附。针对从小仓鼠肾成纤维细胞中纯化的ECM受体制剂的抗体（抗ECMR；Knudsen, K. A., P. E. Rao, C. H. Damsky, and C. A. Buck, 1981, Proc. Natl. Acad. Sci. USA., 78:6071 - 6075）抑制了对LN、FN和Col IV的黏附，但不阻止对其他黏附底物的黏附。除了对黏附的影响外，抗ECMR血清还抑制了PC12细胞和交感神经元在LN底物上的神经突生长。用抗FNR或抗ECMR血清对表面碘化或[3H]葡萄糖胺标记的PC12细胞进行免疫沉淀，在非还原条件下鉴定出三种突出的细胞表面糖蛋白，分子量分别为120、140和180 kD。120-kD糖蛋白可以用32P-正磷酸盐标记，似乎与140-kD和180-kD蛋白非共价结合，与针对禽类整合素复合物β亚基（带3）的抗体发生交叉反应，而整合素复合物本身是ECM成分LN、FN和一些胶原的一种或多种受体。
