Expression and Characterization of Relaxin Family Peptide Receptor 1 Variants

Abstract

G-protein coupled receptors (GPCR) transduce extracellular stimuli into the cell interior and are thus centrally involved in almost all physiological-neuronal processes. This essential function and association with many diseases or pathological conditions explain why GPCRs are one of the priority targets in medical and pharmacological research, including structure determination. Despite enormous experimental efforts over the last decade, both the expression and purification of these membrane proteins remain elusive. This is attributable to specificities of each GPCR subtype and the finding of necessary experimental in vitro conditions, such as expression in heterologous cell systems or with accessory proteins. One of these specific GPCRs is the leucine-rich repeat domain (LRRD) containing GPCR 7 (LGR7), also termed relaxin family peptide receptor 1 (RXFP1). This receptor is characterized by a large extracellular region of around 400 amino acids constituted by several domains, a rare feature among rhodopsin-like (class A) GPCRs. In the present study, we describe the expression and purification of RXFP1, including the design of various constructs suitable for functional/biophysical studies and structure determination. Based on available sequence information, homology models, and modern biochemical and genetic tools, several receptor variations with different purification tags and fusion proteins were prepared and expressed in Sf9 cells (small-scale), followed by an analytic fluorescence-detection size-exclusion chromatography (F-SEC) to evaluate the constructs. The most promising candidates were expressed and purified on a large-scale, accompanied by ligand binding studies using surface plasmon resonance spectroscopy (SPR) and by determination of signaling capacities. The results may support extended studies on RXFP1 receptor constructs serving as targets for small molecule ligand screening or structural elucidation by protein X-ray crystallography or cryo-electron microscopy.

Chemical synthesis of insulin‐like peptide 1 and its potential role in vitellogenesis of the kuruma prawn Marsupenaeus japonicus

“…In contrast, relaxins and INSLs bind to receptors belonging to the G protein-coupled receptor family and regulate various physiological functions, most notably reproduction. 3 The IIS system is also conserved in invertebrates; putative genes encoding ILPs, IRs, and the downstream components are found. For instance, the nematode Caenorhabditis elegans contains 40 ILP genes in its genome, and insulin signaling in this species is thought to regulate aging, stress resistance, larval development, and reproduction.…”

“…The IIS system comprises ligands, binding proteins for ligands, receptors (IR), and various downstream components responsible for intracellular signaling. In contrast, relaxins and INSLs bind to receptors belonging to the G protein‐coupled receptor family and regulate various physiological functions, most notably reproduction 3 …”

Integrate thermostabilized fusion protein apocytochrome b562RIL and N-glycosylation mutations: A novel approach to heterologous expression of human UDP-glucuronosyltransferase (UGT) 2B7

“…The rapid progress of surface plasmon resonance (SPR) spectroscopy is beneficial for studying the ligand-binding to proteins (O'connor et al, 2022) (Speck et al, 2022), which enables the quantitative and real-time measurements of the binding affinity and kinetics of protein-ligand interactions using relatively small amounts of materials, at mediumthroughput (Mizuguchi et al, 2012) (Drozd et al, 2021) (Yin et al, 2020). The dissociation constant (K D ) is commonly obtained using SPR spectroscopy for ligandbinding screening, but few studies have been illustrated for UGTs.…”