Homocysteine effects classical pathway of GPCR down regulation: Galpha(q/11), Galpha(12/13), G(i/o)

Abstract

G protein-coupled receptors (GPCRs) are known to modulate intracellular effectors involved in cardiac 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, beta-arrestin1: ERK activity increases; GRK2 activity increases; beta-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, standardized to ERK1, GRK2, and beta-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 regulation. Hcy was also shown to decrease the content of the following G proteins and other proteins: beta-arrestin1, Galpha(q/11), Galpha(12/13), G(i/o).

Fig. 1

(a) A significant increase of ERK1 activity at 5 and 100 μM Hcy with a standard 60 min time condition (P < 0.05, n = 3). (b) A time-course evaluation of ERK1 activity using 100 μM Hcy and an increasing trend at 15 min and 60 min with statistical significance reached at 30 min (P < 0.05, n = 3)

Fig. 2

(a) A dose-dependent increase in GRK2 activity: trend at 100 μM Hcy, 60 min, significance 500 μM Hcy, 60 min (P < 0.05, n = 3). (b) A time-course evaluation was performed with significance at 100 μM Hcy, 120 min (P < 0.05, n = 3)

Fig. 3

(a) A dose-dependent decrease of p-β -Arrestin1 and β-arrestin1 content: significance at 500 μM Hcy, 60 min (P < 0.05, n = 3). (b) A time-course evaluation was performed showing a significant decrease at 100 μM Hcy 120 min (P < 0.05, n = 3)

Fig. 4

(a) A dose-dependent decrease of p-c-Src and c-Src content: significance at 500 μM Hcy, 60 min (P < 0.05, n = 3). (b) A significant decrease in content of p-c-Src and c-Src with a time-course evaluation of 100 μM Hcy 120 min (P < 0.05, n = 3)

Fig. 5

Hcy treatment showed a dose-dependent decrease of Gα_q/11 at 24 h for the following concentrations: 0, 25, 50, 75, 100, 500 μM; significant decreases were found at 75, 100, and 500 μM (P < 0.05, n = 3). At 48 h, Gα_q/11 content returned to baseline levels for all doses of Hcy except 500 μM where there was a significant decrease (P < 0.05, n = 3)

Fig. 6

Western blot showed no significant increase or decrease of Gα_12/13 for the following concentrations: 0, 25, 50, 75, 100, 500 μM at 24 h (P < 0.05, n = 3). There was a significant decrease of Gα_12/13 using 500 μM Hcy treatment for 48 h (P < 0.05, n = 3)

Fig. 7

Western blot showed no significant increase or decrease of G_i/o for the following concentrations: 0, 25, 50, 75, 100, 500 μM at 24 h (P < 0.05, n = 3). There was a significant decrease of G_i/o using 500 μM Hcy treatment for 48 h (P < 0.05, n = 3)

Fig. 8

Our results suggested Hcy acts as an agonist to potentiate GPCRs and activate the classical GPCR desensitization pathway. We have shown an increase of ERK1 activity that is characteristic of GPCR activation followed by an increase in GRK2 activity and final degradation of β-arrestin1, which is known to occur in down regulation of Class B GPCRs. Moreover, Gα_q/11, Gα_12/13, and G_i/o G proteins that are involved in calcium regulation and attached to GPCRs are down regulated