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 receptor family and is alternatively spliced into at least six different isoforms. The multiple forms are identical in their extracellular and transmembrane regions but differ in lengths. The two predominant isoforms include a long form (OBR(l)) with an intracellular domain of 303 amino acids and a shorter form (OBR(s)) with an intracellular domain of 34 amino acids. We have constructed a recombinant OBR(l) chimera with the green fluorescent protein (GFP) by fusing GFP to the C-terminus of the OBR(l). The OBR(l)-GFP chimera was transiently transfected and expressed in SHSY5Y and HEK293 cells. In a STAT-Luciferase assay we show that the GFP moiety in this chimera did not affect the signalling capacity of OBR(l)-GFP. In both SHSY5Y and HEK293 cells transfected with OBR(l)-GFP, a predominant intracellular green OBR(l)-GFP fluorescence was detected in vesicles also positive for internalized fluorophore conjugated leptin. We also found that treatment with the lysosomotropic reagent monensin did not relocalize OBR(l)-GFP together with the human transferrin receptor in recycling endosomes, indicating OBR(l)-GFP not to participate in this pathway. In biotinylation-streptavidin pulse chase experiments, using antibodies raised against GFP and OBR, we observed that the rate of early appearance of OBR(s) at the cell surface, upon leptin stimulation, was faster than that found for OBR(l)-GFP. Taken together, our results provide novel data concerning the intracellular trafficking of the two different isoforms of the leptin receptor.
Lundin
A
A
Molecular Biology and Genomics, Department of Biology, Pharmacia Corporation, S-112 87, Stockholm, Sweden.
Rondahl
H
H
Walum
E
E
Wilcke
M
M
D019921:Biotinylation; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry; D002460:Cell Line; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry; D008985:Monensin / Q000494:pharmacology; D020033:Protein Isoforms / Q000096:biosynthesis / Q000737:chemistry; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012995:Solubility
D002352:Carrier Proteins; D005456:Fluorescent Dyes; C515448:LEPR protein, human; D008164:Luminescent Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008985:Monensin