Relationship between native and recombinant cholecystokinin receptors: Role of differential glycosylation
Relationship between native and recombinant cholecystokinin receptors: Role of differential glycosylation
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DOI：
10.1097/00006676-199608000-00003
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发表时间：
1996-08-01
期刊：
影响因子：
2.9
通讯作者：
Miller, LJ
中科院分区：
文献类型：
--
作者：
Hadac, EM;Ghanekar, DV;Miller, LJ
为了确定最近克隆的A型胆囊收缩素（CCK）受体cDNA编码的蛋白与大鼠胰腺腺泡细胞上两种不同的质乳蛋白之间的关系，我们建立了一个稳定表达大量该重组蛋白的中国仓鼠卵巢（CHO）细胞系，并使用生化方法对其进行了直接表征。通过亲和标记，该蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶上的迁移速度比先前认为代表最佳候选的M(r) 85,000-95,000分子更快。然而，用内糖苷酶F进行去糖基化分析表明，重组蛋白与原蛋白具有相同大小的核心蛋白，蛋白酶肽图谱进一步支持了这一鉴定，我们推测重组蛋白与原蛋白的结构差异与糖基化差异有关。与此一致的是，凝集素结合实验表明，两者都代表复杂的糖蛋白，但只有天然受体结合的欧洲Ulex凝集素i。由于这种凝集素结合了糖蛋白生物合成后期添加的聚焦残基，因此观察到的不同处理可能只影响了这一步。尽管存在这种结构差异，携带CHO细胞CCK受体的A型CCK受体在功能上与天然腺泡细胞受体无法区分。这包括它启动信号级联的能力，它对稳定的GTP类似物的敏感性，以及它对激动剂和拮抗剂的结合亲和力。该受体表达系统的保真度比天然胰腺腺泡细胞增加了25倍的受体密度，应该为检查该分子内的结构-功能关系提供理想的底物。
In an attempt to establish the relationship between the protein encoded by the recently cloned type A cholecystokinin (CCK) receptor cDNA and the two distinct plasmalemmal proteins on the rat pancreatic acinar cell that were previously described as candidates to represent this receptor, we have established a Chinese hamster ovary (CHO) cell line stably expressing large amounts of this recombinant protein and have used biochemical methods to characterize it directly. Upon affinity labeling, this protein migrated faster on a sodium dodecyl sulfate-polyacrylamide gel than the M(r) 85,000-95,000 molecule previously felt to represent the best candidate. However, deglycosylation with endoglycosidase F demonstrated that it had the same size core protein as that candidate, and this identification was further supported by protease peptide mapping, We postulated that the structural differences between the recombinant and the native proteins related to differences in glycosylation. Consistent with this, lectin-binding experiments demonstrated that both represented complex glycoproteins but that only the native receptor-bound Ulex europeus agglutinin I. Since this lectin binds to fucose residues that are added late in glycoprotein biosynthesis, it is possible that the distinct processing observed affected only that step. In spite of this structural difference, the type A CCK receptor-bearing CHO cell CCK receptor was functionally indistinguishable from the native acinar cell receptor. This included its ability to initiate signaling cascades, its sensitivity to stable GTP analogues, and its binding affinities for agonists and antagonists. The fidelity of this receptor expression system, while representing a 25-fold increase in receptor density over the native pancreatic acinar cell, should provide an ideal substrate for the examination of structure-function relationships within this molecule.