Lokeshwar V B, Fregien N, Bourguignon L Y
Department of Cell Biology and Anatomy, University of Miami School of Medicine, Florida 33101.
J Cell Biol. 1994 Aug;126(4):1099-109. doi: 10.1083/jcb.126.4.1099.
GP85 is one of the most common hemopoietic isoforms of the cell adhesion molecule, CD44. CD44(GP85) is known to contain at least one ankyrin-binding site within its 70 aa cytoplasmic domain and to bind hyaluronic acid (HA) with its extracellular domain. In this study we have mapped the ankyrin-binding domain of CD44(GP85) by deleting various portions of the cytoplasmic region followed by expression of these truncated cDNAs in COS cells. The results of these experiments indicate that the ankyrin-binding domain resides between amino acids 305 and 355. Biochemical analyses, using competition binding assays and a synthetic peptide (NGGNGT-VEDRKPSEL) containing 15 aa between aa 305 and aa 320, support the conclusion that this region is required for ankryin binding. Furthermore, we have constructed a fusion protein in which this 15 aa sequence of CD44(GP85) is transplanted onto another transmembrane protein which does not bind ankyrin. Our results show that this fusion protein acquires the ability to bind ankyrin confirming that the sequence (306NGGNGTVEDRKPSE320L) is a critical part of the ankryin-binding domain of CD44(GP85). In addition, we have demonstrated that deletion of this 15 aa ankyrin-binding sequence from CD44(GP85) results in a drastic reduction (> or = 90%) of HA-binding and HA-mediated cell adhesion. These findings strongly suggest that ankyrin binding to the cytoplasmic domain of CD44(GP85) plays a pivotal role in regulating hyaluronic acid-mediated cell-cell and cell-extracellular matrix interactions.
GP85是细胞黏附分子CD44最常见的造血异构体之一。已知CD44（GP85）在其70个氨基酸的胞质结构域内至少含有一个锚蛋白结合位点，并通过其细胞外结构域结合透明质酸（HA）。在本研究中，我们通过删除胞质区域的不同部分，然后在COS细胞中表达这些截短的cDNA，绘制了CD44（GP85）的锚蛋白结合结构域图谱。这些实验结果表明，锚蛋白结合结构域位于氨基酸305和355之间。生化分析使用竞争结合试验和一个包含305至320位氨基酸之间15个氨基酸的合成肽（NGGNGT-VEDRKPSEL），支持了该区域是锚蛋白结合所必需的这一结论。此外，我们构建了一种融合蛋白，其中将CD44（GP85）的这15个氨基酸序列移植到另一种不结合锚蛋白的跨膜蛋白上。我们的结果表明，这种融合蛋白获得了结合锚蛋白的能力，证实序列（306NGGNGTVEDRKPSE320L）是CD44（GP85）锚蛋白结合结构域的关键部分。此外，我们已经证明，从CD44（GP85）中删除这15个氨基酸的锚蛋白结合序列会导致HA结合和HA介导的细胞黏附急剧减少（≥90%）。这些发现强烈表明，锚蛋白与CD44（GP85）胞质结构域的结合在调节透明质酸介导的细胞间和细胞与细胞外基质相互作用中起关键作用。
