Yang G, Douville P, Gee S, Carbonetto S
Centre for Research in Neurosciences, McGill University, Montreal General Hospital Research Institute, Canada.
J Neurobiol. 1992 Jul;23(5):491-506. doi: 10.1002/neu.480230505.
Laminins are extracellular matrix proteins that mediate their effects on cells through integrin and nonintegrin receptors. Two receptors of 67 and 110 kD that bind laminin with a high affinity (Kd approximately nM) have been reported in neural cells. Here, we discuss these and other nonintegrin laminin receptors that have been implicated in neural function. In addition, we report studies characterizing a 43 kD protein, (P40), immunologically related to the 67 kD laminin receptor, which may be involved in retinal development. In our studies, polyclonal antisera (anti-P-20-A) to a synthetic peptide derived from the sequence of a cDNA for a putative high-affinity laminin receptor (67 kD) detected a protein of 43 kD in immunoblots of adult rat retinas. Immunohistochemistry with this antiserum showed that the retinal immunoreactivity was predominantly localized in the ganglion cell layer of both adult chicken and rat retinas where it appeared to be intracellular. Retinal ganglion cells were shown to be immunoreactive by retrogradely labeling them from the superior colliculus with a lipophilic dye and subsequently with anti-P-20-A antisera. Consistent with the preferential localization of the P-20-A immunoreactivity in ganglion cells, there was a substantial decrease in the amounts of P40 on Western blots following optic nerve section and resulting retinal ganglion cell death. Screening of a rat (PC12 cell) cDNA library with the anti-P-20-A antiserum further confirmed the specificity of the antiserum for the rat homologue of P40. Rat P40 is 97% identical to the mouse and 87% identical to human P40 at the nucleic acid level and 98% at the protein level. Restriction mapping of the rather abundant positive clones in the library that cross-hybridized with a human cDNA probe for P40 indicated that the full-length cDNA of 1.2 kb was the major and perhaps the only cDNA in the library. In Northern blots of adult rat retina, these clones hybridized to a single 1.2-kb transcript. Electroblots of retinal homogenates probed with radioiodinated laminin demonstrated binding to a broad band at 110 kD, but none at 43 kD. Taken together these findings suggest that P40 may not be a laminin receptor and are in keeping with the hydrophilic composition of the protein, its intracellular localization, as well as other features predicted by its nucleic acid sequence.(ABSTRACT TRUNCATED AT 400 WORDS)
层粘连蛋白是细胞外基质蛋白，通过整合素和非整合素受体介导其对细胞的作用。在神经细胞中已报道了两种与层粘连蛋白高亲和力结合（解离常数约为纳摩尔）的67 kD和110 kD受体。在此，我们讨论这些以及其他与神经功能有关的非整合素层粘连蛋白受体。此外，我们报告了对一种43 kD蛋白（P40）的研究，该蛋白与67 kD层粘连蛋白受体在免疫上相关，可能参与视网膜发育。在我们的研究中，针对一种假定的高亲和力层粘连蛋白受体（67 kD）的cDNA序列衍生的合成肽的多克隆抗血清（抗P - 20 - A）在成年大鼠视网膜的免疫印迹中检测到一种43 kD的蛋白。用该抗血清进行免疫组织化学显示，视网膜免疫反应性主要定位于成年鸡和大鼠视网膜的神经节细胞层，且似乎位于细胞内。通过用亲脂性染料从视上丘逆行标记视网膜神经节细胞，随后用抗P - 20 - A抗血清检测，显示神经节细胞具有免疫反应性。与P - 20 - A免疫反应性在神经节细胞中的优先定位一致，视神经切断和随之而来的视网膜神经节细胞死亡后，Western印迹上P40的量大幅减少。用抗P - 20 - A抗血清筛选大鼠（PC12细胞）cDNA文库进一步证实了该抗血清对大鼠P40同源物的特异性。大鼠P40在核酸水平上与小鼠的同源性为97%，与人类的同源性为87%，在蛋白质水平上为98%。文库中与人类P40 cDNA探针交叉杂交的相当丰富的阳性克隆的限制性图谱表明，1.2 kb的全长cDNA是文库中的主要cDNA，可能也是唯一的cDNA。在成年大鼠视网膜的Northern印迹中，这些克隆与一个单一的1.2 kb转录本杂交。用放射性碘化层粘连蛋白探测视网膜匀浆的电印迹显示与110 kD处的一条宽带结合，但在43 kD处无结合。综合这些发现表明，P40可能不是层粘连蛋白受体，这与其蛋白质的亲水性组成、细胞内定位以及其核酸序列预测的其他特征相符。（摘要截短于400字）
