A Requirement for the CD44 Cytoplasmic Domain for Hyaluronan Binding, Pericellular Matrix Assembly, and Receptor-mediated Endocytosis in COS-7 Cells

Abstract CD44-negative COS-7 cells were transfected with expression constructs for CD44H (the predominant CD44 isoform), CD44E (epithelial isoform), or truncation mutant derivatives lacking the carboxyl-terminal 67 amino acids of the cytoplasmic domain, CD44HΔ67 and CD44EΔ67. The truncation mutant CD44HΔ67 is identical to a naturally occurring alternatively spliced “short tail” CD44 isoform (CD44st), which incorporates exon 19 in place of exon 20. CD44st lacks intracellular signaling motifs as well as protein domains necessary for interaction with cytoskeletal components. Transfection of COS-7 cells with each construct yielded equivalent levels of mRNA expression, whereas no CD44 expression was observed in parental, nontransfected COS-7 cells. Western analysis and immunostaining of COS-7 transfectants confirmed CD44 protein expression of the truncation mutant derivatives. COS-7 cells transfected with CD44H or CD44E gained the capacity to bind fluorescein-conjugated HA (fl-HA) and assemble HA-dependent pericellular matrices in the presence of exogenously added HA and proteoglycan. In addition, the CD44H- and CD44E-transfected cells were able to internalize surface-boundfl-HA. COS-7 cells transfected with the vector alone or with either of the mutant CD44 isoforms, CD44HΔ67 or CD44EΔ67, did not exhibit the capacity to assemble pericellular matrices or to bind and internalize the fl-HA. Cotransfection of CD44Δ67 mutants together with CD44H reduced the size of the HA-dependent pericellular matrices. Transfection of bovine articular chondrocytes with CD44Δ67 also inhibited pericellular matrix assembly. Collectively, these results indicate an obligatory requirement for the CD44 receptor cytoplasmic domain for ligand (HA) binding, formation and retention of the pericellular matrix, as well as CD44-mediated endocytosis of HA. In addition, the results suggest a potential regulatory role for the differentially expressed alternatively spliced short tail CD44 isoform.