Lateral mobility of fluorescently labeled G protein subunits in intact cells.

Abstract

Within the overall goal to understand the mechanism of transmembrane signal transduction originating from the $\alpha\sb2$-adrenergic receptor, this thesis has evaluated the collision-coupling model of receptor-effector coupling, and the dissociation model for G protein activation. The G protein subunits $\alpha\sb{\rm o}$ and $\beta\gamma$ were labeled with tetramethylrhodamine (TMR) in vitro, reconstituted into phospholipid vesicles, and incorporated into NG-108-15 neuroblastoma-glioma hybrid cells using polyethylene glycol-mediated cell-vesicle fusion. Prior to reconstitution into vesicles, TMR-$\alpha\sb{\rm o}$ and TMR-$\beta\gamma$ were recombined with their respective unlabeled subunits to yield the complete heterotrimers G protein. The function of exogenous G proteins in the NG-108-15 cells was examined by studying their ability to couple to endogenous $\alpha\sb2$-adrenergic receptors. The subcellular localization of TMR-$\alpha\sb{\rm o}$ and TMR-$\beta\gamma,$ determined using immunoblot analysis and confocal microscopy, indicated the presence of TMR-$\alpha\sb{\rm o}$ in the plasma membrane and in the intracellular region, whereas TMR-$\beta\gamma$ was mainly localized in the plasma membrane. The lateral mobility of TMR-$\alpha\sb{\rm o}$/$\beta\gamma$ and TMR-$\beta\gamma$/$\alpha\sb{\rm o}$ in NG-108-15 cells was measured using the technique of fluorescence photobleaching recovery: the mobile fractions were 32% and 16%, while the diffusion coefficients for the two proteins were 1.2 $\times$ 10$\sp{-9}$cm$\sp2$/sec and 0.8 $\times$ 10$\sp{-9}$cm$\sp2$/sec, respectively. The lateral mobility of a lipid fluorescent probe, 3,3$\sp\prime$-dioctadecylindocarbocyanine iodide (Dil), incorporated into cells by the same method as the fluorescently labeled G protein subunits, had a mobile fraction and diffusion coefficient of 100% and 2.0.0 $\times$ 10$\sp{-9}$cm$\sp2$/sec, respectively. The measured lateral mobility of TMR$\alpha\sb{\rm o}$ and TMR-$\beta\gamma$ is consistent with a model depicting an immobilization of G protein in the plasma membrane with a small fraction of activated $\alpha$ subunits diffusing in the aqueous phase to transmit signals from receptors to effector proteins. The lateral mobility of TMR-$\alpha\sb{\rm o}$ and TMR-$\beta\gamma$ in NG-108-15 cells was not altered by the binding of agonist to the $\alpha\sb2$-adrenergic receptor, or by the cytoskeletal disrupting drugs, cytochalasin D and nocodazole. These results indicate that small fractions of G proteins are activated by ligand-bound receptors and that neither actin nor tubulin are involved in anchoring TMR-$\beta\gamma.$.