Homocysteine effects classical pathway of GPCR down regulation

G protein-coupled receptors (GPCRs) are known to modulate intracellular effectors involved in car- diac function. We recently reported homocysteine (Hcy)- induced ERK-phosphorylation was suppressed by pertussis toxin (PTX), which suggested the involvement of GPCRs in initiating signal transduction. An activated GPCR undergoes down regulation via a known mechanism involving ERK, GRK2, b-arrestin1: ERK activity increa- ses; GRK2 activity increases; b-arrestin1 is degraded. We hypothesized that Hcy treatment leads to GPCR activation and down regulation. Microvascular endothelial cells were treated with Hcy. Expression of phospho-ERK1 and phospho-GRK2 was determined using Western blot, stan- dardized to ERK1, GRK2, and b-actin. Hcy was shown to dephosphorylate GRK2, thereby enhancing the activity. The results provided further evidence that Hcy acts as an agonist to activate GPCRs, followed by their down regu- lation. Hcy was also shown to decrease the content of the following G proteins and other proteins: b-arrestin1, Gaq/11 ,G a12/13 ,G i/o.