Expression and intracellular localization of leptin receptor long isoform-GFP chimera

The leptin receptor (OBR) and its ligand leptin (OB) are key players in the regulation of body weight. The OBR is a member of the class I cytokine rec eptor family and is alternatively spliced into at least six different isofo rms. The multiple forms are identical in their extracellular and transmembr ane regions but differ in lengths. The two predominant isoforms include a l ong form (OBRl) with an intracellular domain of 303 amino acids and a short er form (OBRs) with an intracellular domain of 34 amino acids. We have cons tructed a recombinant OBRl chimera with the green fluorescent protein (GFP) by fusing GFP to the C-terminus of the OBRl. The OBRl-GFP chimera was tran siently transfected and expressed in SHSY5Y and HEK293 cells. In a STAT-Luc iferase assay we show that the GFP moiety in this chimera did not affect th e signalling capacity of OBRl-GFP. In both SHSY5Y and HEK293 cells transfec ted with OBRl-GFP, a predominant intracellular green OBRl-GFP fluorescence was detected in vesicles also positive for internalized fluorophore conjuga ted leptin. We also found that treatment with the lysosomotropic reagent mo nensin did not relocalize OBRl-GFP together with the human transferrin rece ptor in recycling endosomes, indicating OBRl-GFP not to participate in this pathway. In biotinylation-streptavidin pulse chase experiments, using anti bodies raised against GFP and OBR, we observed that the rate of early appea rance of OBRs at the cell surface, upon leptin stimulation, was faster than that found for OBRl-GFP. Taken together, our results provide novel data co ncerning the intracellular trafficking of the two different isoforms of the leptin receptor. (C) 2000 Elsevier Science B.V. All rights reserved.