Disease-associated GPR56 Mutations Cause Bilateral Frontoparietal Polymicrogyria via Multiple Mechanisms

Nien-Yi Chiang,Cheng-Chih Hsiao,Yi-Shu Huang,Hsin-Yi Chen,I-Ju Hsieh,Gin-Wen Chang,Hsi-Hsien Lin

doi:10.1074/jbc.m110.183830

Abstract

Loss-of-function mutations in the gene encoding G protein-coupled receptor 56 (GPR56) lead to bilateral frontoparietal polymicrogyria (BFPP), an autosomal recessive disorder affecting brain development. The GPR56 receptor is a member of the adhesion-GPCR family characterized by the chimeric composition of a long ectodomain (ECD), a GPCR proteolysis site (GPS), and a seven-pass transmembrane (7TM) moiety. Interestingly, all identified BFPP-associated missense mutations are located within the extracellular region of GPR56 including the ECD, GPS, and the extracellular loops of 7TM. In the present study, a detailed molecular and functional analysis of the wild-type GPR56 and BFPP-associated point mutants shows that individual GPR56 mutants most likely cause BFPP via different combination of multiple mechanisms. These include reduced surface receptor expression, loss of GPS proteolysis, reduced receptor shedding, inability to interact with a novel protein ligand, and differential distribution of the 7TM moiety in lipid rafts. These results provide novel insights into the cellular functions of GPR56 receptor and reveal molecular mechanisms whereby GPR56 mutations induce BFPP.

Highlights

One G protein-coupled receptors (GPCRs) of interest known to be involved in diverse biological systems is G proteincoupled receptor 56 (GPR56)
Through the analysis of the wild type (WT) and the bilateral frontoparietal polymicrogyria (BFPP)-associated GPR56 point mutants, we identify functional characteristics of GPR56 that might provide a molecular explanation for the disorder: 1) stringent requirement for efficient cell surface expression; 2) GPCR proteolysis site (GPS) cleavage-independent shedding of GPR56; 3) Identification of a novel protein ligand that does not interact with the N-terminal BFPP mutants; 4) GPR56-ligand interaction promotes cell adhesion; 5) Localization of the 7TM subunit to membrane lipid rafts
BFPP-associated GPR56 Mutant Proteins Are Deficient in Intracellular Trafficking, Cell Surface Expression and/or GPS Proteolytic Cleavage—To investigate the effect of BFPP-associated point mutations on the expression and GPS proteolytic modification of GPR56, WT, and mutant GPR56 proteins were tagged with a hemagglutinin (HA) and c-Myc epitope at the N and C terminus, respectively (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—General reagents were obtained from Sigma-Aldrich, unless otherwise specified. Cells were incubated for 1 h with fluorochrome-conjugated goat anti-mouse IgG in blocking buffer (1:200). For the Dynabead-cell binding assay, 25 ␮l of protein A or G-conjugated paramagnetic Dynabeads௡ (Dynals; Lake Success NY) were washed three times in 0.1% BSA/PBS for 5 min each. GPR56-mFc protein (5 ␮g/reaction) was mixed with Dynabeads for 1 h, washed to remove unbound proteins, and incubated with cells in 0.1% BSA/PBS (1 ϫ 106 cells/ml; total volume, 0.5 ml) on ice for 1 h with constant rotation. Lipid Raft Separation—All procedures are carried out on ice. Cells (ϳ5 ϫ 106 cells) were washed three times with ice-cold PBS, lysed in 150 ␮l ice-cold TNET buffer (25 mM Tris/ HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10 mM NaF, 1 mM sodium pyrophosphate, 1 mM Na3VO4 and 1ϫ Protease inhibitor mixture) for 30 min. Seven equal fractions (ϳ600 ␮l/fraction) were collected from the top of the tube

RESULTS

HeLa ϩϩϩ

DISCUSSION