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.
Highlights
In 1926, Frederick Hisaw set the starting point for relaxin research when he transferred blood plasma from a pregnant to a virgin guinea pig, thereby triggering pubic ligament relaxation in the recipient animal (Bylander et al, 1987)
Known and postulated from the receptor sequence, RXFP1 consists of a transmembrane helix domains (TMD) typical of G-protein coupled receptors (GPCR), comprising seven helices and six helix-interconnecting loops (Figure 1)
The extracellular part comprises an low-density lipoprotein class A (LDLa) domain, a linker region between the LDLa and the leucine-rich repeat domain (LRRD), which is connected via a hinge region to TM1
Summary
In 1926, Frederick Hisaw set the starting point for relaxin research when he transferred blood plasma from a pregnant to a virgin guinea pig, thereby triggering pubic ligament relaxation in the recipient animal (Bylander et al, 1987). The corresponding relaxin binding partner was identified (Hsu et al, 2000; Hsu et al, 2002). Based on sequence similarities with previously discovered leucine-rich repeat containing receptors (LGRs), this relaxin receptor was originally termed LGR7 but later renamed RXFP1 (Braun et al, 1991; Hsu et al, 1998; Bathgate et al, 2006). Receptor or ligand knock-out led to an age-dependent increase in tissue fibrosis, whereby relaxin was studied extensively as an anti-fibrotic therapeutic (Samuel et al, 2003; Samuel et al, 2004a; Samuel et al, 2004b; McBride et al, 2017; Samuel et al, 2017)
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