Grove B D, Bowditch R, Gordon T, del Zoppo G, Ginsberg M H
Department of Cellular Biology and Anatomy, Louisiana State University Medical Center, Shreveport 71130.
Anat Rec. 1994 Jul;239(3):231-42. doi: 10.1002/ar.1092390302.
Gravin, a novel, high molecular weight, intracellular protein, is expressed in endothelial cells and several other adherent cell types in vitro. To gain insights into its function, we examined the distribution of gravin in tissues.
Affinity-purified polyclonal and monoclonal antibodies were raised against a bacterial fusion protein corresponding to the carboxyl terminus of gravin and against affinity-isolated gravin. The specificity of the antibodies was characterized by immunoblotting bacterial, cell, and tissue extracts. The characterized antibodies were used to localize gravin in baboon tissue sections by immunocytochemistry and immunofluorescence microscopy.
The antibodies specifically immunoblotted the fusion protein and recognized either a band at 250 kDa or a doublet at 300 kDa on immunoblots of MG63 cells, HEL cells stimulated with phorbol ester, and several baboon tissues. In tissue sections, cell types that express gravin included fibroblasts, components of the peripheral and central nervous system, the adrenal medulla, the somatic layer of Bowman's capsule, cells associated with the glomerulus, and smooth muscle of certain organs. In contrast, most epithelia and all endothelia, with the exception of endothelia of the hepatic sinusoids and intestinal lacteals, lacked gravin. Levels of gravin mRNA expression in stimulated HEL cells increased dramatically when cells were stimulated in the presence of cycloheximide, suggesting that gravin expression may be partly regulated by protein-dependent mRNA catabolism.
These data indicate that gravin expression is regulated in endothelial cells, possibly through protein-dependent mRNA catabolism. The strong expression of gravin in fibroblasts, neurons, and cells derived from neural crest in vivo and in adherent cells in vitro further suggests that this protein may play role in the modulation of cell motility and adhesion.
Gravin是一种新型的高分子量细胞内蛋白，在体外内皮细胞和其他几种贴壁细胞类型中表达。为深入了解其功能，我们检测了gravin在组织中的分布。
针对与gravin羧基末端相对应的细菌融合蛋白以及亲和分离的gravin制备亲和纯化的多克隆和单克隆抗体。通过免疫印迹法检测细菌、细胞和组织提取物来鉴定抗体的特异性。将鉴定后的抗体用于通过免疫细胞化学和免疫荧光显微镜在狒狒组织切片中定位gravin。
这些抗体特异性地免疫印迹融合蛋白，并在MG63细胞、佛波酯刺激的HEL细胞以及几种狒狒组织的免疫印迹上识别出250 kDa的条带或300 kDa的双峰。在组织切片中，表达gravin的细胞类型包括成纤维细胞、外周和中枢神经系统的成分、肾上腺髓质、鲍曼囊的体细胞层、与肾小球相关的细胞以及某些器官的平滑肌。相比之下，除肝血窦内皮细胞和肠乳糜管内皮细胞外，大多数上皮细胞和所有内皮细胞均缺乏gravin。当在放线菌酮存在的情况下刺激细胞时，刺激后的HEL细胞中gravin mRNA表达水平显著增加，这表明gravin表达可能部分受蛋白质依赖性mRNA分解代谢的调节。
这些数据表明gravin的表达在内皮细胞中受到调节，可能是通过蛋白质依赖性mRNA分解代谢。gravin在体内的成纤维细胞、神经元和神经嵴来源的细胞以及体外贴壁细胞中的强表达进一步表明该蛋白可能在细胞运动和黏附的调节中发挥作用。
