SAT-043 A Trafficking Defective Mutant PROKR2, Identified In Patients With Hypogonadotropic Hypogonadism, Cycles Between Endoplasmic Reticulum (ER) And Golgi To Attenuate ER Stress

Abstract

G protein-coupled receptors (GPCRs) are the largest family of transmembrane proteins and are a major class of therapeutic drug targets. Mutations in GPCRs resulting in loss of function or alteration of signaling can cause inherited or acquired diseases. Such mutations frequently cause impaired cell surface expression, resulting from protein misfolding and intracellular trapping due to careful monitoring by the cellular protein surveillance system. However, protein quality control mechanisms and intracellular trafficking of misfolded GPCRs remain incompletely understood. Here, we investigated the effects of mutations in prokineticin receptor 2 (PROKR2), identified in patients with hypogonadotropic hypogonadism or Kallmann syndrome, characterized by GnRH deficiency, as a model of the GPCR trafficking process. We first confirmed the impairment in cellular localization and downstream signaling of these PROKR2 mutants. To better understand the mechanisms underlying differences in cellular pathway targeting between wild type (WT) PROKR2 and P290S PROKR2, a trafficking defective mutant in which the mutation is in one of the most highly conserved amino acids among GPCRs, located in the 6th transmembrane domain, we performed a comparative interactome study using immunoprecipitation followed by mass spectrometry analysis. This revealed that P290S PROKR2 interacts strongly with proteins involved in the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway, transport to the Golgi, and retrograde transport from the Golgi to the ER. Guided by these interactome findings, we demonstrated that both the trafficking defective mutant, P290S PROKR2, as well as WT PROKR2 were degraded by the ERAD pathway, with the P290S mutant entering the ERAD pathway to a greater extent instead of being transported to the plasma membrane. Surprisingly, we found that P290S PROKR2 enters the Golgi, even though it is degraded by the ERAD pathway from the ER. When misfolded proteins accumulate in the ER, they evoke ER stress, which activates the unfolded protein response and can cause cell death. We found that misfolded P290S PROKR2 cycles to the ER-Golgi interface to reduce ER stress and to promote efficient degradation via the ERAD pathway. Our studies demonstrate that misfolded GPCRs are controlled in the early secretory pathway to maintain cellular homeostasis, thereby providing a fundamental strategy for restoration of defective cell surface transport caused by GPCR mutations as well as protection from the initiation and progression of human disease caused by ER stress. Our findings offer insights which could lead to development of potential therapeutic targets for diseases caused by human GPCR mutations.