Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells

Abstract

G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3′-5′-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2) and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third intracellular loop in the regulation of GPR3 activity.

Towards a better understanding of the cannabinoid-related orphan receptors GPR3, GPR6, and GPR12

“…In contrast, Lowther and coworkers suggested that GPR3 does not signal following endocytosis (Lowther et al 2011; Lowther et al 2013). They observed that GPR3 signals at the cell surface being susceptible to desensitization by a β-arrestin2 and GRK2 (G protein-coupled receptor kinase)-mediated mechanism.…”

“…They observed that GPR3 signals at the cell surface being susceptible to desensitization by a β-arrestin2 and GRK2 (G protein-coupled receptor kinase)-mediated mechanism. In fact, endocytic inhibition increased cAMP levels and GPR3 accumulation at the cell-surface, whereas overexpression of GRK2 and β-arrestin2 led to lower cAMP levels and a reduced GPR3 cell-surface expression (Lowther et al 2013). …”

GPR3, GPR6, and GPR12 as novel molecular targets: their biological functions and interaction with cannabidiol

“…Forskolin stimulation maximally stimulates cAMP production such that G s -mediated production is nonsignificant and any constitutive coupling to G i becomes apparent by inhibition of cAMP signaling. Constitutive inhibition of [11,[20][21][22] ↑ AC activation [11,20] ↑ cAMP reporter [21,22] Constitutively active [20,22] ↑ AC activation [20] ↑ cAMP reporter [22] Constitutively active [20][21][22] ↑ AC activation [20] ↑ cAMP reporter [21,22] G i Constitutively active [20] PTX treatment ↑AC activation [20] G αi overexpression ↓ cAMP [20] No effect on cAMP reporter after forskolin stimulation [22] Constitutively active [20] PTX treatment ↑AC activation [20] G αi overexpression ↓ cAMP [20] PTX treatment ↓ ligand-induced Ca 2+ mobilization [17] ↓ cAMP reporter after forskolin stimulation [22] Constitutively active [22] ↓ cAMP reporter after forskolin stimulation [22] PTX treatment ↓ ligand-induced GIRK currents [19] Non-G protein ERK1/2 pathway Signals via ERK1/2 and Akt [23] Signals via ERK1/2 and sphingosine kinase phosphorylation [17] Signals via ERK1/2 [24] β-arrestin2 No ligand-induced β-arrestin2 recruitment [28] Internalization and desensitization using GRK2 and β-arrestin2 [25] Modulation of Aβ production via β-arrestin2 [26,27] No ligand-induced β-arrestin2 recruitment [28] No ligand-induced β-arre...…”

Hunting for the function of orphan GPCRs – beyond the search for the endogenous ligand

“…Some of the remaining orphan 7TM proteins (more than 100) might have ligand‐independent functions, expressed as a significant degree of constitutive activity triggering functional responses in the absence of ligands (Figure B). The molecular explanation for constitutive activity is often based on specific receptor sequences that stabilize the active receptor conformation, thus allowing the interaction with various proteins that induce cellular signalling events (Lowther et al ., ).…”

“…The degree of constitutive activity of an orphan 7TM protein not only depends on its expression level, but also on regulatory proteins such as β‐arrestin 2 and the GPCR kinase 2 that may be implicated in receptor desensitization as shown for GPR3 (Lowther et al ., ). An interesting example is GPR17, which has two constitutively active splice variants, GPR17‐L and GPR17‐S, in humans that are expressed in a tissue‐specific manner and that differ in the length of their N‐termini and activate Gα i proteins independently from the putative GPR17 ligands, the leukotrienes, LTC 4 and LTD 4 (Benned‐Jensen and Rosenkilde, ).…”