Constitutive Gαi Coupling Activity of Very Large G Protein-coupled Receptor 1 (VLGR1) and Its Regulation by PDZD7 Protein

Qiao-Xia Hu,Jun-Hong Dong,Hai-Bo Du,Dao-Lai Zhang,Hong-Ze Ren,Ming-Liang Ma,Yuan Cai,Tong-Chao Zhao,Xiao-Lei Yin,Xiao Yu,Tian Xue,Zhi-Gang Xu,Jin-Peng Sun

doi:10.1074/jbc.m114.549816

Abstract

The very large G protein-coupled receptor 1 (VLGR1) is a core component in inner ear hair cell development. Mutations in the vlgr1 gene cause Usher syndrome, the symptoms of which include congenital hearing loss and progressive retinitis pigmentosa. However, the mechanism of VLGR1-regulated intracellular signaling and its role in Usher syndrome remain elusive. Here, we show that VLGR1 is processed into two fragments after autocleavage at the G protein-coupled receptor proteolytic site. The cleaved VLGR1 β-subunit constitutively inhibited adenylate cyclase (AC) activity through Gαi coupling. Co-expression of the Gαiq chimera with the VLGR1 β-subunit changed its activity to the phospholipase C/nuclear factor of activated T cells signaling pathway, which demonstrates the Gαi protein coupling specificity of this subunit. An R6002A mutation in intracellular loop 2 of VLGR1 abolished Gαi coupling, but the pathogenic VLGR1 Y6236fsx1 mutant showed increased AC inhibition. Furthermore, overexpression of another Usher syndrome protein, PDZD7, decreased the AC inhibition of the VLGR1 β-subunit but showed no effect on the VLGR1 Y6236fsx1 mutant. Taken together, we identified an independent Gαi signaling pathway of the VLGR1 β-subunit and its regulatory mechanisms that may have a role in the development of Usher syndrome.

Highlights

The signaling and regulatory mechanism of the orphan receptor very large G protein-coupled receptor 1 (VLGR1) remains elusive
To test whether VLGR1 can be processed into two fragments in a manner similar to other adhesion GPCRs, we made a construct of the C-terminal portion of VLGR1 that contained the intact GAIN domain, the seven-transmembrane region, and the intracellular C-terminal tail (Fig. 1A)
Similar to the chemical cleavage that occurs at the GPCR proteolytic site (GPS) of other adhesion GPCRs, the autocleavage of VLGR1 can be accelerated by the strong nucleophile NH2OH (Fig. 1E) (19)

Summary

Introduction

The signaling and regulatory mechanism of the orphan receptor VLGR1 remains elusive. Results: The cleaved VLGR1 ␤-subunit constitutively coupled to G␣i and was regulated by the VLGR1 ␣-subunit, a diseaseassociated mutation, and PDZD7. Co-expression of the G␣iq chimera with the VLGR1 ␤-subunit changed its activity to the phospholipase C/nuclear factor of activated T cells signaling pathway, which demonstrates the G␣i protein coupling specificity of this subunit. Overexpression of another Usher syndrome protein, PDZD7, decreased the AC inhibition of the VLGR1 ␤-subunit but showed no effect on the VLGR1 Y6236fsx1 mutant.

Results

Conclusion