Identification and Characterization of Distinct C-Terminal Domains of the Human Hydroxycarboxylic Acid Receptor-2 That Are Essential for Receptor Export, Constitutive Activity, Desensitization, and Internalization

Abstract

The human hydroxycarboxylic acid receptor 2 (HCA 2 ), also known as GPR109A and HM74a, was first identified as a niacin receptor and has recently received significant attention because of its potential to clinically modify plasma lipids in a favorable manner. Our recent studies have demonstrated that the niacin-induced internalization of HCA 2 receptors is regulated by G protein-coupled receptor kinase (GRK) 2 and arrestin3 and that internalized receptors rapidly recycle back to the cell surface. The investigation presented here used a combination of amino acid deletion and site-directed mutagenesis to identify structural and functional domains within the HCA 2 C terminus and explore their potential roles in receptor phosphorylation, desensitization, and internalization. We first constructed four mutants with deletions of 10 to 15 amino acids each that were distinct from truncated mutants. We successfully identified different domains responsible for receptor export, constitutive activity, desensitization, phosphorylation, and internalization. We also generated a comprehensive series of alanine substitution mutants, replacing conserved serine and threonine residues in the C terminus with alanine residues to pinpoint the key residues that are essential for GRK2-mediated phosphorylation and arrestin3 association. Moreover, we found that a sequence from residues 329 to 343 in the C-terminal tail of HCA 2 plays a crucial role in keeping HCA 2 in an inactive conformation. These data demonstrate the importance of distinct domains within the C terminus of HCA 2 for receptor cell surface expression, desensitization, and internalization and phosphorylation and stabilization of an inactive receptor conformation.

Nicotinic acid timed to feeding reverses tissue lipid accumulation and improves glucose control in obese Zucker rats[S]

“…An insulin-NiAc synergy is not just a theoretical possibility but was in fact seen in our previous hyperinsulinemic clamp studies (10). Although and insulin are mediated via adipocyte cAMP lowering, but their combined effects are theoretically synergistic with NiAc, decreasing cAMP formation (28), and insulin, ligand-induced GPR109A desensitization and internalization) (34). Second, excessive exposures can invoke additional complicating mechanisms beyond FFA lowering.…”

Coupling Metastasis to pH-Sensing GPR68 Using a Novel Small Molecule Inhibitor

“…Given the apparent association rs61745752 with cancer metastasis, we sought to determine functional consequence of the variant which causes a truncation after amino acid 335 (E336X). Mutations in the c-terminal tail of GPCRs can have a number of consequences including loss of desensitization, and inactivation (Li et al, 2012) . GPCR desensitization is modulated through beta-arrestin binding to domains that are defined by GIRK phosphorylation (Ahn et al, 2003;Luttrell and Lefkowitz, 2002;Sente et al, 2018) .…”

Distinct regions in the C-Terminus required for GLP-1R cell surface expression, activity and internalisation

“…Using a number of C-terminal deletion mutants of the hGLP-1R, this study determined residues 411e418 are critical for newly synthesised hGLP-1R cell surface expression. The membrane proximal region of the C-terminal domain is important for the biosynthetic trafficking of many GPCRs such as a 2B -AR, AT 2 R type 1A and dopamine receptor 1 (D1R) to the plasma membrane (Li et al, 2012). The membrane proximal region of the C-terminal domain of the a 2B -AR and AT 2 R type 1A (Duvernay et al, 2004) have been shown to be essential for exportation of the receptor from the ER.…”