Nuclear Localization of Protease-Activated Receptor 2 Dictates Angiogenesis

Abstract

Purpose: : Neurons govern angiogenesis in the developing and ischemic retina. Protease-activated receptor 2 (PAR2) contributes to the development of new blood vessels and is abundantly expressed in retinal ganglion cells (RGCs). We detected PAR2 at the cell nucleus of these neurons. To date, many G-protein coupled receptors (GPCRs) have been reported at the cell nucleus where they evoke in situ gene induction. However, the sub-cellular origin of nuclear GPCRs, the mechanisms governing this localization and their nuclear function, as well as the in vivo physiologic manifestation of nuclear GPCRs are not known.

Methods: : Nuclear localization of PAR2 was confirmed by electron microscopy. Live confocal imaging and flow cytometry analysis were used to assess translocation of PAR2 to the cell nucleus. PAR2 mutants were generated by PCR cloning. Interaction of PAR2 with proteins and transcription factors was ascertained by co-immunoprecipitation, confocal imaging, as well as MS/MS, ChiP assay, and EMSA respectively. Intra-vitreal injections of lentivirus-containing silencing shRNA and PAR2 mutants were expressed in retinas of wild type and PAR2 knockout mice.

Results: : Here, we show that PAR2 translocates from the plasma membrane to the cell nucleus, requiring specific receptor domains (C-terminus and nuclear localization signals) as well as the recruitment of Importin-β1 and Sorting nexin 11 (SNX11), which interact with microtubules. In turn, nuclear PAR2 recruits transcription factor Sp1 to trigger angiogenic genes and ensued neovascularization.

Conclusions: : This is the first report describing the in vivo physiologic manifestation governed by the nuclear localization of a receptor, notably in this case regulation of angiogenesis, and unprecedented mechanisms of gene induction of a GPCR (PAR2) at the cell nucleus. GPCRs are the largest family of therapeutic drug targets; findings infer a need to target a given receptor based on its cellular localization.

Keywords: neovascularization • ganglion cells • receptors: pharmacology/physiology