Abstract
Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B(4) receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory G(i2) protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human alpha(5) integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB(4) binding in the presence of the purified G protein G alpha(i2). The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5'-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified G alpha(i2) beta(1) gamma(2) protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal.
Highlights
Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear
This approach is based first on the fusion of the receptor to an integrin fragment that allowed efficient overexpression in metal affinity chromatography; LTB4, leukotriene B4; PLC, phospholipase C; PurF, glutamine phosphoribosyl pyrophosphatase amidotransferase; SEC, size exclusion chromatography; V2, arginine-vasopressin receptor subtype 2; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate; LTB4-568, LTB4 labeled with Alexa Fluor-568; Ni-NTA, nickel-nitrilotriacetic acid; DPC, n-dodecyl phosphocholine; HDM, hexadecyl--D-maltoside; CHS, cholesteryl hemisuccinate; MOPS, 4-morpholinepropanesulfonic acid; GST, glutathione S-transferase; OTR, oxytocin receptor
Identification of New Fusion Partners for Efficient G protein-coupled receptors (GPCRs) Overexpression in E. coli inclusion bodies (IBs)—To produce recombinant GPCRs in sufficient amounts to reconstitute, in vitro receptor-G protein complexes, we used an approach that had been initially described for the olfactory OR5 receptor and is based on accumulation of the receptor target in E. coli IBs as a fusion protein and on subsequent in vitro refolding [29, 30]
Summary
The construction, expression, and purification of the different GPCR fusions, as well as their thrombin cleavage and the subsequent purification of isolated GPCRs are described in the supplemental data. The antagonist was removed from the eluted protein by dialysis for 36 h against buffer containing 12.5 mM sodium borate, pH 7.5, 10 mM NaCl and DPC/HDM/asolectin/CHS Following this step, the BLT2 concentration was in the 10Ϫ6–10Ϫ7 M range. For measuring systematic activation of the G protein, the basal rate of GTP␥S binding was determined by monitoring the relative increase in the intrinsic fluorescence (exc ϭ 300 nm, em ϭ 345 nm) of G␣i2 (200 nM of purified G␣i2) in the presence of BLT2 (20 nM, the final receptor to G protein molar ratio is 1:10) in buffer containing 10 mM MOPS, pH 7.2, 130 mM NaCl, and 2 mM MgCl2 for 40 min at 15 °C after the addition of 10 M GTP␥S. Statistical significance of the differences between independent groups was assessed by paired t test
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