HYALURONIC ACID-INDUCED LYMPHOCYTE SIGNAL-TRANSDUCTION AND HA RECEPTOR (GP85 CD44)-CYTOSKELETON INTERACTION/

The purposes of this study are to characterize the binding of hyaluron ic acid (HA) to mouse T lymphoma cells, to measure changes in intracel lular Ca2+ after HA binding, to elucidate the interaction between the HA receptor, GP85(CD44), and ankyrin in the membrane skeleton, and fin ally to correlate these events with HA receptor patching/capping and c ell adhesion to HA. First, we established an in vivo assay using [H-3] HA to measure the binding of HA to mouse T lymphoma cells, and found t hat the binding of [H-3]HA to these cells is readily inhibited by the addition of anti-GP85(CD44) antibody suggesting that GP85(CD44) is the HA receptor. Next, we examined various signal transducing events that occur after HA binds to its receptor on mouse T lymphoma cells. The r esults of these studies indicate that the concentration of intracellul ar Ca2+ (as measured by Fura-2 fluorescence) begins to increase within seconds, and reaches a maximal level 5 min after the addition of HA t o the cells. After this increase of intracellular Ca2-, HA induces bot h its receptors, GP85(CD44), to form patched/capped structures, and ce ll adhesion to HA-coated plates. Furthermore, we have determined that GP85(CD44) binds directly and specifically to ankyrin (K(d) almost-equ al-to 1.94 nM) in a saturable manner; and that ankyrin is preferential ly accumulated underneath the HA-induced GP85(CD44) capped structures. The Ca2+ ionophore, ionomycin, was found to stimulate HA-induced rece ptor capping and adhesion while EGTA (a Ca2+ chelator), nefedipine/bep ridil (Ca2+ channel blockers), W-7 (a calmodulin antagonist), and cyto chalasin D (a microfilament inhibitor), but not colchicine (a microtub ule disrupting agent), inhibit HA-induced receptor redistribution and adhesion to HA-coated plates. These findings strongly suggest that ank yrin plays an important role in linking the HA receptor, GP85(CD44), t o the membrane-associated actomyosin contractile system during hyaluro nic acid-mediated lymphocyte activation.