Polar Expression and Phosphorylation of Human Leptin Receptor Isoforms in Paired, Syncytial, Microvillous and Basal Membranes from Human Term Placenta

Abstract

The hormone leptin (OB) and its receptor (OB-R) are key homeostatic regulators of mammalian body weight. Two predominant isoforms of OB-R are expressed by alternative splicing: the long form, OB-RL, with full signalling capacity is highly expressed in the hypothalamus and the short, signalling-defective form, OB-Rs, is ubiquitously expressed.In a previous study we detected expression of OB-RL and OB-Rs in human syncytiotrophoblast cells using in situ hybridization and immunohistochemistry (Bodner et al., 1999).The aim of this study was to investigate leptin receptor isoform expression and phosphorylation in paired, syncytial, microvillous and basal membranes from human term placenta by Western blot analysis. Both the OB-RL and the OB-Rs isoforms were detected in the syncytial membrane preparations. The OB-RL isoform was observed exclusively in microvillous membranes, whereas the OB-Rs isoform was found in both microvillous and basal membrane preparations. No significant differences were observed between syncytial membranes from normal and type 1 diabetic pregnancies. To test the phosphorylation capacity of the OB-R isoforms, microvillous and basal membrane vesicles loaded with ATP were stimulated with leptin and the phosphorylation status of the OB-R at the tyrosine 985 (Y985) was determined. A single band at the molecular weight corresponding to the molecular weight of the OB-RL isoform was detected exclusively in the ATP-loaded microvillous vesicles.We conclude that the long form OB-RL is expressed exclusively in the microvillous membrane of the syncytiotrophoblast and is capable of being phosphorylated, suggesting that it has signal transduction capacity.