Heterologous expression of functional G‐protein‐coupled receptors inCaenorhabditis elegans

Abstract

New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.

Inherent Instability of the Retinitis Pigmentosa P23H Mutant Opsin

“…WT and P23H Bovine Rhodopsin Constructs and Generation of TG Worm Lines-For the pan-neuronal bovine opsin ((b)opsin) expression construct (Genscript, Piscataway, NJ), codonoptimized (32) (b)opsin cDNA (sequence reference for (b)opsin, NP_001014890) was driven by the H20 promoter in a pBluscript KS(ϩ) vector (28,30,33). The pan-neuronal bovine opsin P23H expression construct itself was generated from WT (b)opsin vector by site-directed mutagenesis.…”

“…This procedure was modified from that previously reported for WT isorhodopsin (28). Briefly, TG worms were cultured for two generations (5-6 days) in 10 150-mm high growth medium (HGM) culture dishes seeded with HB101 Escherichia coli bacteria and then transferred into a 10-liter fermenter filled with autoclaved S medium (31).…”

“…1B) together with a DNA vector encoding DsRed fluorescent protein under the same promoter. Selective genomic integration was indicated by DsRed fluorescence in the nervous system (28,30), and the TG worm line with the greatest P23H opsin expression was used for this study. Previously described TG C. elegans expressing WT bovine opsin (WT worms) were used as controls (28,30).…”

“…Selective genomic integration was indicated by DsRed fluorescence in the nervous system (28,30), and the TG worm line with the greatest P23H opsin expression was used for this study. Previously described TG C. elegans expressing WT bovine opsin (WT worms) were used as controls (28,30). As quantified by quantitative RT-PCR, expression of WT opsin and P23H opsin in the two TG worm lines was comparable at the mRNA level ( Fig.…”

“…4, A and B). Biochemical characterization of P23H isorhodopsin was performed immediately after purification, whereas WT isorhodopsin was analyzed either promptly after purification or after storage at Ϫ80°C (28,30). No biochemical changes were noted in WT isorhodopsin after weeks at Ϫ80°C.…”