The purposes of this study are to characterize the binding of hyaluronic acid (HA) to mouse T lymphoma cells, to measure changes in intracellular Ca2+ after HA binding, to elucidate the interaction between the HA receptor, GP85(CD44), and ankyrin in the membrane skeleton, and finally to correlate these events with HA receptor patching/capping and cell adhesion to HA. First, we established an in vivo assay using [3H]HA to measure the binding of HA to mouse T lymphoma cells, and found that the binding of [3H]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 results of these studies indicate that the concentration of intracellular Ca2+ (as measured by Fura-2 fluorescence) begins to increase within seconds, and reaches a maximal level 5 min after the addition of HA to the cells. After this increase of intracellular Ca2+, HA induces both its receptors, GP85(CD44), to form patched/capped structures, and cell adhesion to HA-coated plates. Furthermore, we have determined that GP85(CD44) binds directly and specifically to ankyrin (Kd ≈ 1.94 nM) in a saturable manner; and that ankyrin is preferentially accumulated underneath the HA-induced GP85(CD44) capped structures. The Ca2+ ionophore, ionomycin, was found to stimulate HA-induced receptor capping and adhesion while EGTA (a Ca2+ chelator), nefedipine/bepridil (Ca2+ channel blockers), W-7 (a calmodulin antagonist), and cytochalasin D (a microfilament inhibitor), but not colchicine (a microtubule disrupting agent), inhibit HA-induced receptor redistribution and adhesion to HA-coated plates. These findings strongly suggest that ankyrin plays an important role in linking the HA receptor, GP85(CD44), to the membrane-associated actomyosin contractile system during hyaluronic acid-mediated lymphocyte activation.
|Original language||English (US)|
|Number of pages||11|
|Journal||Journal of Immunology|
|State||Published - 1993|
ASJC Scopus subject areas
- Immunology and Allergy