Addition of a Carboxy-Terminal Green Fluorescent Protein Does Not Alter the Binding and Signaling Properties of Relaxin Family Peptide Receptor 3

摘要

The relaxin family peptide receptor 3 (RXFP3) is the cognate receptor for the neu-ropeptide 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 un-tagged 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.