TY - JOUR
T1 - Ankyrin-binding domain of CD44(GP85) is required for the expression of hyaluronic acid-mediated adhesion function
AU - Lokeshwar, Vinata B.
AU - Fregien, Nevis
AU - Bourguignon, Lilly Y.W.
PY - 1994/8
Y1 - 1994/8
N2 - 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 (NGGNGTVEDRKPSEL) containing 15 aa between aa 305 and aa 320, support the conclusion that this region is required for ankyrin 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 ankyrin- 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 (≥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.
AB - 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 (NGGNGTVEDRKPSEL) containing 15 aa between aa 305 and aa 320, support the conclusion that this region is required for ankyrin 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 ankyrin- 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 (≥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.
UR - http://www.scopus.com/inward/record.url?scp=0028141591&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028141591&partnerID=8YFLogxK
U2 - 10.1083/jcb.126.4.1099
DO - 10.1083/jcb.126.4.1099
M3 - Article
C2 - 7519619
AN - SCOPUS:0028141591
SN - 0021-9525
VL - 126
SP - 1099
EP - 1109
JO - Journal of Cell Biology
JF - Journal of Cell Biology
IS - 4
ER -