ANKYRIN-BINDING DOMAIN OF CD44(GP85) IS REQUIRED FOR THE EXPRESSION OF HYALURONIC ACID-MEDIATED ADHESION FUNCTION

GP85 is one of the most common hemopoietic isoforms of the cell adhesi on molecule, CD44. CD44(GP85) is known to contain at least one ankyrin -binding site within its 70 aa cytoplasmic domain and to bind hyaluron ic acid (HA) with its extracellular domain. In this study we have mapp ed the ankyrin-binding domain of CD44(GP85) by deleting various portio ns of the cytoplasmic region followed by expression of these truncated cDNAs in COS cells. The results of these experiments indicate that th e ankyrin-binding domain resides between amino acids 305 and 355. Bioc hemical analyses, using competition binding assays and a synthetic pep tide (NGGNGTVEDRKPSEL) containing 15 aa between aa 305 and aa 320, sup port 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 prot ein which does not bind ankyrin. Our results show that this fusion pro tein acquires the ability to bind ankyrin confirming that the sequence ((306)NGGNGTVEDRKPSE(320)L) 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 d rastic reduction (greater than or equal to 90%) of HA-binding and HA-m ediated cell adhesion. These findings strongly suggest that ankyrin bi nding to the cytoplasmic domain of CD44(GP85) plays a pivotal role in regulating hyaluronic acid-mediated cell-cell and cell-extracellular m atrix interactions.