SAT-LB137 Rescue of Misfolded G-Protein Coupled Estrogen Receptor, GPER, from the Endoplasmic Reticulum via Natural and Synthetic Estrogens

GPER bears structural and functional characteristics shared by members of the G-protein coupled receptor (GPCR) superfamily, the largest class of cell surface receptors, with more than 800 members encoded in the human genome. GPER is localized predominately in intracellular membranes, in many but not all cell types, and its surface expression is modulated by steroid hormones and during tissue homeostasis. An intracellular staining pattern is not unique among GPCRs, which deploy a diverse array of posttranslational regulatory mechanisms to determine cell surface expression, effectively regulating cognate ligand binding and activity. Here, we show nascent GPER undergoes strict quality control via endoplasmic reticulum associated degradation (ERAD) requiring direct poly-ubiquitinylation of GPER and valosin-containing protein VCP/p97-mediated segregation of misfolded proteins from the ER membrane to the cytoplasm for delivery to the 26S proteasome. Specifically, we find that inhibition of p97 using the pharmacological compound, CB-5083, or by doxycycline-inducible p97 shRNA results in the accumulation of immature glycosylated GPER in the ER. Inhibition of proteasome function facilitates anterograde trafficking with the transport of nonfunctional GPER to the plasma membrane as indicated by no increase in specific estrogen binding using (3)H-17β-estradiol in a radioreceptor assay. The forward trafficking of misfolded GPER requires transit through the Golgi as treatment with brefeldin A (BFA) prevents GPER plasma membrane expression. Substitution of all three lysines (K333, K342, and K357) encoded in the cytoplasmic tail of GPER with arginines blunts its polyubiquitinylation and allows GPER to evade degradation by quality control but does not result in increased plasma membrane expression suggesting that additional structural motifs encoded within GPER control its anterograde trafficking. In contrast, functional GPER is recovered at the plasma membrane of human SKBR3 breast cancer cells treated with either 17β-estradiol or the GPER selective antagonist, G15, in the presence of cycloheximide resulting in increased surface GPER. Thus, our findings suggest that estrogens, both natural and synthetic, can function as pharmacochaperones capable of promoting the correct folding of GPER and enhanced expression of functional GPER at the plasma membrane.