Addition of a carboxy-terminal green fluorescent protein does not alter the binding and signaling properties of relaxin family Peptide receptor 3.
The relaxin family peptide receptor 3 (RXFP3) is the cognate receptor for the neuropeptide relaxin-3. RXFP3 was tagged at the carboxy-terminus with a variant of the green fluorescent protein (GFP(2)) for use in receptor localization studies. RXFP3-GFP(2) was examined to ensure it retained binding and signaling properties similar to untagged RXFP3. Competition for [(125)I]INSL5/H3 relaxin chimera binding to RXFP3 and RXFP3-GFP(2) indicated that the carboxy-terminal tag did not affect receptor binding or receptor internalization. RXFP3-GFP(2) activated ERK1/2 with a similar potency to RXFP3 when transiently expressed in CHO-K1 or HEK293T cells, suggesting that the GFP(2) tag did not affect receptor function. This study demonstrated that addition of a carboxy-terminal fusion protein to RXFP3 did not alter the binding or signaling properties of RXFP3, making RXFP3-GFP(2) a useful tool for future receptor localization and trafficking studies.
Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Clayton, Victoria 3800, Australia.
Emma T
ET
van der Westhuizen
John D
JD
Wade
Patrick M
PM
Sexton
Roger J
RJ
Summers
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012065:Relaxin / Q000494:pharmacology; D015398:Signal Transduction
D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins; C410092:RXFP3 protein, human; D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins; D012065:Relaxin; D000242:Cyclic AMP; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3