Functional Characterization of CALCRL (rs369317777) in IPSC-Derived Endothelial Cells

Abstract

Introduction: The calcitonin receptor-like receptor (CLR) is a G protein-coupled receptor involved in various physiological processes. Its function depends on its interaction with receptor activity-modifying proteins (RAMPs). When CLR associates with RAMP1, it responds to calcitonin gene-related peptide, a pain transmitter and vasodilator. When combined with RAMP2 or RAMP3, it functions as an adrenomedullin receptor and is involved in vasodilation, growth promotion, and hormone secretion regulation. Hence, the CLR/RAMP complexes are crucial for cardiovascular physiology and pain transmission. Knowledge Gap: While significant progress has been made in understanding the canonical functions of the CLR, limited research has focused on investigating the impact of naturally occurring variations on its molecular and physiological roles. Addressing this gap, our collaboration with the Maurice Wilkins Centre has revealed a coding variant (rs369317777) in the CALCRL gene, prevalent in individuals of Polynesian descent but rare in other ethnic groups. This variant results in the substitution of a conserved isoleucine residue with threonine at position 446 (446T) in the receptor's C-terminal tail. Preliminary evidence suggests that this variant may contribute to blood pressure regulation. However, a comprehensive exploration of its unique molecular mechanisms is currently lacking. Aims and hypothesis: Hence, this study sought to utilize human induced pluripotent stem cell (iPSC)-derived endothelial cell (EC) as a model for in vitro investigation of the 446T variant's role in blood pressure regulation. Aims: 1) employ CRISPR-Cas9 gene editing to introduce the 446T CALCRL variant into iPSCs; 2) differentiate the gene-edited iPSCs into ECs to evaluate the impact of CLR ligands on iPSC-EC activity; 3) assess cellular activities such as NO production, growth, migration, and cAMP production, which are mediated by CLR/RAMP signalling pathways. It is proposed that the variant CLR may trigger a hyperactivated effect upon binding to its ligands, potentially leading to an extensive vasodilatory effect. Key results/Conclusions: In this study, we generated three iPSC lines, including WT, knock-in (446T variant), and knock-out lines, providing valuable tools for studying vascular biology. By differentiating these iPSCs into iPSC-ECs, we established a cellular model and found that the 446T variant reduced efficacy in ADM-induced cAMP production, potentially affecting Gαs coupling and receptor pathways. These findings lay the foundation for further investigations, including more experiments, validation from additional iPSC lines and primary ECs, and evaluation of Gαs- and Gαq-mediated pathways.