A Tyrosine Residue on the TSH Receptor Stabilizes Multimer Formation

Rauf Latif,Syed Ahmed Morshed,Terry F Davies,Krzysztof Michalek,Anna Carla Goldberg

doi:10.1371/journal.pone.0009449

Abstract

BackgroundThe thyrotropin stimulating hormone receptor (TSHR) is a G protein coupled receptor (GPCR) with a large ectodomain. The ligand, TSH, acting via this receptor regulates thyroid growth and thyroid hormone production and secretion. The TSH receptor (TSHR) undergoes complex post –translational modifications including intramolecular cleavage and receptor multimerization. Since monomeric and multimeric receptors coexist in cells, understanding the functional role of just the TSHR multimers is difficult. Therefore, to help understand the physiological significance of receptor multimerization, it will be necessary to abrogate multimer formation, which requires identifying the ectodomain and endodomain interaction sites on the TSHR. Here, we have examined the contribution of the ectodomain to constitutive multimerization of the TSHR and determined the possible residue(s) that may be involved in this interaction.Methodology/Principal FindingsWe studied ectodomain multimer formation by expressing the extracellular domain of the TSHR linked to a glycophosphotidyl (GPI) anchor in both stable and transient expression systems. Using co-immunoprecipitation and FRET of tagged receptors, we established that the TSH receptor ectodomain was capable of multimerization even when totally devoid of the transmembrane domain. Further, we studied the effect of two residues that likely made critical contact points in this interaction. We showed that a conserved tyrosine residue (Y116) on the convex surface of the LRR3 was a critical residue in ectodomain multimer formation since mutation of this residue to serine totally abrogated ectodomain multimers. This abrogation was not seen with the mutation of cysteine 176 on the inner side of the LRR5, demonstrating that inter-receptor disulfide bonding was not involved in ectodomain multimer formation. Additionally, the Y116 mutation in the intact wild type receptor enhanced receptor degradation.Conclusions/SignificanceThese data establish the TSH receptor ectodomain as one site of multimerization, independent of the transmembrane region, and that this interaction was primarily via a conserved tyrosine residue in LRR3.

Highlights

The thyroid stimulating hormone receptor (TSHR), a typical 7transmembrane G protein coupled receptor (GPCR), on the surface of thyrocytes, is the master regulator of thyroid growth and development
A control mutation, isoleucine 117 (I117), in the outer loop of LLR3 adjacent to Y116 did not lead to any increased constitutive receptor degradation. (Figure 7C).The cAMP signaling capability of Y116 receptor, was not affected by the enhanced degradation of the receptor forms (Figure 7D) suggesting ectodomain degradation was post-signaling. Glycoprotein hormone receptors such as the LHR, FSHR and thyrotropin stimulating hormone receptor (TSHR) belonging to Class A receptors exist as dimeric and higher order forms in native and transfected cells [13,14,15,32,33,34] the physiological relevance of these receptor forms is still under investigation
While it is not uncommon for DTT to have nonspecific effects on the dissociation of whole receptor complexes as observed with the P2Y12 and b2AR GPCRs [35,36], the reduction of higher complexes by DTT suggested that ectodomain oligomer formation may be mediated, in part, by disulfide bond formation via the several cysteine residues found on the ectodomain of the TSHR [1,6]

Summary

Introduction

The thyroid stimulating hormone receptor (TSHR), a typical 7transmembrane GPCR, on the surface of thyrocytes, is the master regulator of thyroid growth and development. TSH acting via TSHR regulates thyroid hormone production and secretion. The TSHR consists of a large extracellular ectodomain of 415 residues inclusive of a signal peptide of 21aa. The thyrotropin stimulating hormone receptor (TSHR) is a G protein coupled receptor (GPCR) with a large ectodomain. The ligand, TSH, acting via this receptor regulates thyroid growth and thyroid hormone production and secretion. The TSH receptor (TSHR) undergoes complex post –translational modifications including intramolecular cleavage and receptor multimerization. To help understand the physiological significance of receptor multimerization, it will be necessary to abrogate multimer formation, which requires identifying the ectodomain and endodomain interaction sites on the TSHR. We have examined the contribution of the ectodomain to constitutive multimerization of the TSHR and determined the possible residue(s) that may be involved in this interaction

Methods

Results

Conclusion