The major objective of this proposal is to describe in molecular terms the mechanism by which the binding of a mitogenic polypeptide (growth factor) to its plasma membrane receptor generates an intracellular response. The particular response selected for these studies is the rapid activation of the Na+/H+ antiporter in the plasma membrane upon addition of growth factors to cells. The antiporter activity will be assayed by 22Na+ flux measurements as well as by determination of growth factor-induced elevation of the intracellular pH (pHi) which is a consequence of the activation of Na+/H+ exchange. New potent inhibitors of Na+/H+ exchange from the amiloride series labeled with radioactive isotopes will be used to quantitate the antiporter and follow it during purification procedures. Studies of the mechanism of activation of the Na+/H+ antiporter will focus on the role of growth factor receptor-associated protein kinase. Phosphorylation will be assayed by a variety of techniques, including the use of antibodies raised against a purified antiporter as well as an Na+/H+ exchange deficient mutant recently developed in Pouyssegur's laboratory. Both direct and """"""""cascade"""""""" phosphorylation mechanisms will be examined. If successful, these studies would shed light on the molecular mechanisms that are involved in the regulation of normal and possibly malignant cell growth. In a related project, the role of the stimulation of Na+/H+ exchange in the mitogenic response will be examined. This question will be addressed by developing inhibitors from the amiloride series bearing a fully charged group which, unlike amiloride analogs previously tested, should be relatively nontoxic and thus suitable for studies of the physiological role of the Na+/H+ antiporter.
