Identification and characterization of a crucial guanine nucleotide exchange factor (GEF), p63RhoGEF in blood pressure regulation in zebrafish aiming for a novel high-throughput drug screening.

Abstract

Smooth muscle contraction incorporates two pathways: the intracellular Ca2+ concentration-dependent and Ca2+ sensitization pathways. For years, the effector between the key players of the Ca2+ sensitization pathway, G protein-coupled receptors and a small GTPase, RhoA, was unknown until we identified a Guanine nucleotide Exchange Factor (GEF), p63RhoGEF. Unlike RhoA, ubiquitously expressed and thus irrelevant to a drug target with tissue specificity, p63RhoGEF expression is limited to smooth muscle. Our goal is to identify compounds that specifically inhibit p63RhoGEF, blood vessel constriction, and thus hypertension. We showed p63RhoGEF being activated upon microvasculature constriction in mice. However, using mice is not cost-efficient in large-scale inhibitory compound screening. Therefore, we moved our focus to zebrafish, one of the model animals of vertebrates, to invent a fast, easy-to-use, and non-invasive methodology of screening. We can directly observe the fin vascular network while the animal is alive, i.e., in physiologically ideal conditions. The bioinformatical analysis identified two pairs of potential p63RhoGEF isoforms in zebrafish. While reporting one Pfu amplified isoform, we identified some mutations in two other Taq amplified isoforms compared to those in the NCBI database and nailed the most relevant candidates through bioinformatical analysis. We report the process of analysis and the sequences of all amplicons. We constructed expression plasmids, over-expressed those p63RhoGEF candidates, and acquired preliminary observations. In the future, we will not only pursue functional analysis but also in situ hybridization to locate the expression of those isoforms to show that those newly identified isoforms are useful targets for drug screening. The system proposed here must set a solid foundation for screening p63RhoGEF inhibitors in physiologically ideal conditions and in a swift, cost-efficient manner.