Abstract
Mechanisms that prevent aggregation and promote folding of nascent G protein-coupled receptors (GPCRs) remain poorly understood. We identified chaperonin containing TCP-1 subunit eta (CCT7) as an interacting partner of the β-isoform of thromboxane A2 receptor (TPβ) by yeast two-hybrid screening. CCT7 coimmunoprecipitated with overexpressed TPβ and β2-adrenergic receptor (β2AR) in HEK 293 cells, but also with endogenous β2AR. CCT7 depletion by small interfering RNA reduced total and cell-surface expression of both receptors and caused redistribution of the receptors to juxtanuclear aggresomes, significantly more so for TPβ than β2AR. Interestingly, Hsp90 coimmunoprecipitated with β2AR but virtually not with TPβ, indicating that nascent GPCRs can adopt alternative folding pathways. In vitro pull-down assays showed that both receptors can interact directly with CCT7 through their third intracellular loops and C-termini. We demonstrate that Trp334 in the TPβ C-terminus is critical for the CCT7 interaction and plays an important role in TPβ maturation and cell-surface expression. Of note, introducing a tryptophan in the corresponding position of the TPα isoform confers the CCT7-binding and maturation properties of TPβ. We show that an interaction with a subunit of the CCT/TCP-1 ring complex (TRiC) chaperonin complex is involved in regulating aggregation of nascent GPCRs and in promoting their proper maturation and expression.