Determination of protein-bound palmitate turnover rates using a three-compartment model that formally incorporates [3H]palmitate recycling.

The observation that the palmitoylation state of certain proteins can be biologically modulated led to the proposal that it could, much like phosphorylation, be an important dynamic regulator of protein function. However, based on single-phase exponential decay analysis of data from [(3)H]palmitate pulse/chase experiments, the measured protein-bound palmitate turnover rates were often found to be too slow to account for rapid physiological responses. This paper reports that exponential decay does not adequately describe the results of such experiments because it fails to account for the recycling of [(3)H]palmitate from cellular lipids to palmitoyl CoA. Taking this recycling into account, a three-compartment model was used to deduce the time-dependent changes of cellular [(3)H]palmitoyl CoA and to infer the time course for the incorporation of [(3)H]palmitate into proteins. The validity of the inferences made by the model was checked against data obtained by metabolic labeling of endogenous HEK293 cell proteins. In addition, the model could account for reported anomalies, discrepancies, and apparently paradoxical observations obtained by traditional analysis of data from pulse/chase experiments. Including the recycling of cellular palmitate in the formal description of the system offers a new tool for quantitative assessment of protein-bound palmitate turnover rates. Through the re-evaluation of these rates, the model provides a means for the reassessment of the potential physiological implications of dynamic palmitoylation. The model may also be generally applicable to other areas of research where recycling of tracer is a concern.

Phosphorylation-independent desensitization of GABA(B) receptor by GRK4.

Agonist-promoted desensitization of the heterodimeric metabotropic GABA(B) receptor was investigated. Whereas no desensitization was observed in HEK293 cells heterologously expressing the receptor, GABA and the synthetic agonist baclofen induced a robust desensitization in cerebellar granule cells endogenously expressing the receptor. Taking advantage of this cell-specific desensitization phenotype, we identified GRK4 as the kinase involved in the neuronal desensitization. Transfection of small interference RNA directed against GRK4 significantly reduced GRK4 levels in cerebellar granule cells and strongly inhibited the agonist-promoted desensitization. Reciprocally, transfection of GRK4 in HEK293 cells restored agonist-promoted desensitization, confirming that this kinase is sufficient to support desensitization. Surprisingly, this desensitization occurred in the absence of ligand-induced receptor phosphorylation and could be promoted by GRK4 mutants deleted of their kinase domain. Taken together, these results suggest that GRK4 plays a central role in the agonist-promoted desensitization of GABA(B) receptor and that it does so through an atypical mechanism that challenges the generally accepted model linking the kinase activity of GRKs to their role in receptor desensitization.

Role of palmitoylation/depalmitoylation reactions in G-protein-coupled receptor function.

G-protein-coupled receptors (GPCRs) constitute one of the largest protein families in the human genome. They are subject to numerous post-translational modifications, including palmitoylation. This review highlights the dynamic nature of palmitoylation and its role in GPCR expression and function. The palmitoylation of other proteins involved in GPCR signaling, such as G-proteins, regulators of G-protein signaling, and G-protein-coupled receptor kinases, is also discussed.