O'Brien D A, Gabel C A, Rockett D L, Eddy E M
Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Endocrinology. 1989 Dec;125(6):2973-84. doi: 10.1210/endo-125-6-2973.
Mannose 6-phosphate (Man 6-P) receptors participate in the targeting of both newly synthesized and extracellular acid hydrolases to lysosomes, and also may mediate the effects of a number of growth factors. In this study, Man 6-P receptors were isolated from [35S]methionine-labeled germ cells and Sertoli cells by phosphomannan-Sepharose affinity chromatography and were analyzed by polyacrylamide gel electrophoresis and autoradiography. Mouse pachytene spermatocytes and round spermatids isolated by unit gravity sedimentation synthesized predominantly the 46,000 mol wt (Mr) cation-dependent Man 6-P receptor and only low levels of the 270,000 Mr cation-independent Man 6-P receptor. In contrast, Sertoli cells synthesized substantial amounts of the cation-independent Man 6-P receptor, but little of the cation-dependent receptor. To determine if these receptors function on the cell surface, we have monitored Man 6-P receptor-mediated endocytosis in isolated germ cells and Sertoli cells. In pachytene spermatocytes and round spermatids, [125I]Man 6-P-bearing ligands were internalized and processed to lower Mr forms by an endocytic mechanism that was time dependent, proportional to cell number, and inhibited by Man 6-P. Like germ cells, Sertoli cells in primary culture endocytosed radiolabeled Man 6-P-bearing ligands at levels that were about 10% of the endocytic activity measured for 3T3 fibroblasts. This low endocytic activity correlates with the levels of the cation-independent Man 6-P receptor synthesized by germ cells, but not with the higher levels synthesized by Sertoli cells. Membrane binding assays verified the high steady state levels of the cation-independent Man 6-P receptor in Sertoli cells, suggesting that the low endocytic activity detected in these cells may result from restricted expression of the cation-independent receptor on the cell surface. These results indicate that both spermatogenic and Sertoli cells have surface Man P-6 receptors capable of mediating endocytosis. However, these cells exhibit marked differences in the expression of the cation-independent and -dependent Man 6-P receptors, perhaps reflecting differential roles of these receptors in protein trafficking and/or intercellular communication during germ cell differentiation.
甘露糖6 - 磷酸（Man 6 - P）受体参与将新合成的和细胞外酸性水解酶靶向运输到溶酶体，并且还可能介导多种生长因子的作用。在本研究中，通过磷酸甘露聚糖 - 琼脂糖亲和层析从[35S]甲硫氨酸标记的生殖细胞和支持细胞中分离出Man 6 - P受体，并通过聚丙烯酰胺凝胶电泳和放射自显影进行分析。通过单位重力沉降分离的小鼠粗线期精母细胞和圆形精子细胞主要合成46,000道尔顿分子量（Mr）的阳离子依赖性Man 6 - P受体，而仅合成少量270,000 Mr的阳离子非依赖性Man 6 - P受体。相比之下，支持细胞合成大量的阳离子非依赖性Man 6 - P受体，但很少合成阳离子依赖性受体。为了确定这些受体是否在细胞表面发挥作用，我们监测了分离的生殖细胞和支持细胞中Man 6 - P受体介导的内吞作用。在粗线期精母细胞和圆形精子细胞中，携带[125I]Man 6 - P的配体通过一种时间依赖性、与细胞数量成比例且受Man 6 - P抑制的内吞机制被内化并加工成较低Mr形式。与生殖细胞一样，原代培养的支持细胞内吞放射性标记的携带Man 6 - P的配体，其水平约为3T3成纤维细胞内吞活性的10%。这种低内吞活性与生殖细胞合成的阳离子非依赖性Man 6 - P受体水平相关，但与支持细胞合成的较高水平无关。膜结合试验证实了支持细胞中阳离子非依赖性Man 6 - P受体的高稳态水平，表明在这些细胞中检测到的低内吞活性可能是由于阳离子非依赖性受体在细胞表面的表达受限所致。这些结果表明，生精细胞和支持细胞都具有能够介导内吞作用的表面Man P - 6受体。然而，这些细胞在阳离子非依赖性和依赖性Man 6 - P受体的表达上表现出明显差异，这可能反映了这些受体在生殖细胞分化过程中蛋白质运输和/或细胞间通讯中的不同作用。
