Naturally occurring HCA1 missense mutations result in loss of function: potential impact on lipid deposition

Jamie R Doyle,Alan S Kopin,Jacqueline M Lane,Martin Beinborn

doi:10.1194/jlr.m034660

Abstract

The hydroxy-carboxylic acid receptor (HCA1) is a G protein-coupled receptor that is highly expressed on adipocytes and considered a potential target for the treatment of dyslipidemia. In the current study, we investigated the pharmacological properties of naturally occurring variants in this receptor (H43Q, A110V, S172L, and D253H). After transient expression of these receptors into human embryonic kidney 293 cells, basal and ligand-induced signaling were assessed using luciferase reporter gene assays. The A110V, S172L, and D253 variants showed reduced basal activity; the S172L mutant displayed a decrease in potency to the endogenous ligand L-lactate. Both the S172L and D253H variants also showed impaired cell surface expression, which may in part explain the reduced activity of these receptors. The impact of a loss in HCA1 function on lipid accumulation was investigated in the adipocyte cell line, OP9. In these cells, endogenous HCA1 transcript levels rapidly increased and reached maximal levels 3 days after the addition of differentiation media. Knockdown of HCA1 using siRNA resulted in an increase in lipid accumulation as assessed by quantification of Nile Red staining and TLC analysis. Our data suggest that lipid homeostasis may be altered in carriers of selected HCA1 missense variants.

Highlights

The hydroxy-carboxylic acid receptor (HCA1) is a G protein-coupled receptor that is highly expressed on adipocytes and considered a potential target for the treatment of dyslipidemia
From the NHLBI Exome Sequencing Project (ESP) Exome Variant Server, a database of single-nucleotide polymorphisms (SNPs) assembled based on whole-exome sequencing, we selected three additional variants to be included in our study: A110V, S172L, and D253H
Frequencies of the A110V, S172L, and D253H variants were obtained from the NHLBI ESP Exome Variant Server [Exome Variant Server, NHLBI Exome Sequencing Project (ESP), Seattle, WA (URL: http://evs.gs. washington.edu/EVS/)]

Summary

Introduction

The hydroxy-carboxylic acid receptor (HCA1) is a G protein-coupled receptor that is highly expressed on adipocytes and considered a potential target for the treatment of dyslipidemia. We found that each of the variants examined, with the exception of the H43Q isoform, showed some degree of loss of function in assays assessing basal activity, ligand induced signaling, and/or surface expression. Cells were transiently transfected in serum-free media using PEI (0.1 μl/well of a 1 mg/ml solution) [15, 16] with cDNAs encoding i) wild-type or mutant HCA1 (or pcDNA1.1, the empty expression vector), ii) an SRE-luciferase reporter gene (SRE5×luc), iii) Gq5i66V, a chimeric G␣q protein with five carboxyl-terminal amino acids corresponding to G␣i and valine substituted for glycine at amino acid position 66, and iv) a ␤-galactosidase control to enable correction of interwell variability.

Results

Conclusion