The overall goal of this proposal is to evaluate the organization of hormone receptors of sites of interaction in the plane of the plasma membrane to gain a more detail understanding of the mechanisms of hormonally-induced transmembrane signalling. The location of steroid and catecholamine receptors in the plane of the freeze-fractured plasma membrane will be evaluated by a new electron microscopic autoradiographic technique, monolayer freeze-fracture autoradiography (MONOFARG). Given the resolution data obtained from test systems included as part of this proposal, MONOFARG will be used to determine the distribution of radioisotopically labelled molecules in the plane of both intact and split membrane bilayers. It will thus be possible to determine if radioisotope is associated with regions that contain integral membrane polypeptides (intramembranous particles) or with smooth areas thought to be predominantly lipid.
The specific aims of this proposal are several. First, the autoradiographic sensitivity and resolution limits for MONOFARG will be established for radioiodinated, tritiated, and carbon-14 test systems. Second, the distribution of radiolabeled lipids and polypeptides in the plane of the split membrane will be evaluated using the human erythrocyte membrane as a model. The in-plane distribution of both tritiated cholesterol (prototypic for lipophilic hormone studies) and radioiodinated integral membrane polypeptides (for putative transmembrane hormone receptors) will be examined. In addition, it will be possible for the first time to determine the lipid and protein composition of freeze-fractured intramembranous particles in unfixed and chemically unmodified membranes physically stabilized by freezing. Given this background the in-plane distribution of tritiated estrogen and of radioiodinated adrenergic receptors will be evaluated. The long-term objectives of this research are to examine functionally and pathologically revelant biomembrane systems by MONOFARG. These projects will share the common goal of understanding the molecular organization and mechanisms of transmembrane processes that include both signalling and permeability in plasma membranes derived from normal and diseased cells.
