Geranylgeranylation facilitates proteasomal degradation of rho G-proteins in human trabecular meshwork cells.

Purpose: To determine the role of posttranslational isoprenylation in regulating Rho G-protein activation and stability in human trabecular meshwork (TM) cells.

Methods: Transformed human TM cells (GTM3) were incubated for 24 hours in the presence of activated lovastatin (10 μM) to enhance the endogenous synthesis of latent Rho proteins. Medium was replaced, cycloheximide (CHX) was added to inhibit synthesis of new proteins, and lovastatin-pretreated cells were subsequently incubated (0-24 hours) in the absence (control) or presence of farnesyl pyrophosphate (10 μM) or geranylgeranyl pyrophosphate (10 μM). Relative changes in the content of total and GTP-bound Rho G-proteins were quantified by Western immunoblot and GTP-binding ELISA, respectively. Changes in filamentous actin stress fiber organization were visualized with AlexaFluor488-conjugated phalloidin.

Results: GTM3 cells cultured in the presence of lovastatin exhibited a loss of actin stress fiber organization concomitant with a marked accumulation of cytosolic inactive (GDP-bound) Rho G-proteins. Addition of geranylgeranyl pyrophosphate to the culture medium restored actin stress fiber organization while selectively facilitating the subcellular redistribution of accumulated Rho proteins from cytosol to membrane and increasing RhoA activation. Geranylgeranyl pyrophosphate selectively enhanced the degradation of newly synthesized Rho proteins. Epoxomicin, a potent and selective inhibitor of the 20S proteasome, prevented geranylgeranyl-enhanced degradation of Rho proteins.

Conclusions: Posttranslational geranylgeranylation selectively alters the lifecycle of newly synthesized Rho proteins by facilitating their membrane translocation, functional activation, and turnover. Geranylgeranylation represents a novel mechanism by which active Rho proteins are targeted to the proteasome for degradation in human TM cells.