Endospanins Regulate a Postinternalization Step of the Leptin Receptor Endocytic Pathway

Abstract

Endospanin-1 is a negative regulator of the cell surface expression of leptin receptor (OB-R), and endospanin-2 is a homologue of unknown function. We investigated the mechanism for endospanin-1 action in regulating OB-R cell surface expression. Here we show that endospanin-1 and -2 are small integral membrane proteins that localize in endosomes and the trans-Golgi network. Antibody uptake experiments showed that both endospanins are transported to the plasma membrane and then internalized into early endosomes but do not recycle back to the trans-Golgi network. Overexpression of endospanin-1 or endospanin-2 led to a decrease of OB-R cell surface expression, whereas shRNA-mediated depletion of each protein increased OB-R cell surface expression. This increased cell surface expression was not observed with OB-Ra mutants defective in endocytosis or with transferrin and EGF receptors. Endospanin-1 or endospanin-2 depletion did not change the internalization rate of OB-Ra but slowed down its lysosomal degradation. Thus, both endospanins are regulators of postinternalization membrane traffic of the endocytic pathway of OB-R.Leptin and the leptin receptor are key proteins in pathways regulating energy balance and body weight in both humans and rodents (1-3). The leptin receptor is a single membrane-spanning protein of the class I cytokine receptor family. The leptin receptor gene is transcribed in a number of different messenger RNAs by alternative promoter usage and mRNA splicing (4). Most of these transcripts are splice variants that encode receptor isoforms with different cytoplasmic tails, which are referred to as OB-Ra, OB-Rc, OB-Rd (short isoforms), and OB-Rb (long isoform). The long splice variant OB-Rb mediates the majority of the effects of leptin, mainly via the JAK-STAT signaling pathway (1, 5, 6). The exact function of the short isoforms is still unclear. Short and long isoforms follow very similar intracellular membrane trafficking pathways (7-9).Recently, we identified a negative regulator of leptin receptor function (10). This protein, originally named leptin receptor gene-related protein, or OB-RGRP, is encoded by a transcript expressed from the leptin receptor gene (11). The OB-RGRP mRNA shares two exons that are untranslated in leptin receptor transcripts and contains two additional exons that are absent in leptin receptor transcripts. This transcript is named LEPROT (leptin receptor overlapping transcript). This overlapping gene structure is conserved in humans and rodents (11,12). We named this protein endospanin-1, due to its topology and intracellular localization. We have shown that endospanin-1 down-regulates leptin signaling by lowering the expression of the receptor at the cell surface and thus the sensitivity of the cell to leptin. Furthermore, the physiological relevance of endospanin-1 as a regulator of OB-R function was evaluated in mice. The expression of a lentivirus-delivered short hairpin RNA (shRNA) directed against endospanin-1 in the arcuate nucleus of the hypothalamus prevented the...

Increased Expression of Fibroblast Growth Factor 21 (FGF21) during Chronic Undernutrition Causes Growth Hormone Insensitivity in Chondrocytes by Inducing Leptin Receptor Overlapping Transcript (LEPROT) and Leptin Receptor Overlapping Transcript-like 1 (LEPROTL1) Expression

“…Silencing of LEPROT increases leptin receptor localization on the plasma membrane (20,22), whereas overexpression of LEPROT decreases cell-surface exposure of leptin receptor (20) and GH receptor (19). The effect of LEPROT on the intracellular trafficking of membrane receptors appear to be specific, as it has been shown that it does not affect EGF receptor trafficking (19,20,22). LEPROT and LEPROTL1 mRNAs are widely expressed in mammalian tissues and organs, including the liver.…”

“…The leptin receptor overlapping transcript (LEPROT, also known as endospanin-1 or obesity receptor gene-related protein, OB-RGRP) belongs to a family of genes that includes two other members: one in mammalian cells (LEPROT-like 1, LEPROTL1, or endospanin-2) (20,24) and another in Saccharomyces cerevisiae (vacuolar protein sorting 55, VPS55) (25). These genes encode small proteins with four potential transmembrane domains.…”

“…At the end of the incubation, the cultures were washed 5 times with ice-cold PBS containing 0.2% BSA (PBS/BSA), detached with trypsin/ethylenediamine tetraacetate for cell counting, and solubilized in 0.5 mol/liter NaOH and 0.1% Triton X-100 for radioactivity measurement. To determine whether the effects of LEPROT and/or LEPROTL1 on GH binding were specific, we measured EGF binding, as it has been previously shown that the EGF receptor intracellular processing is not affected by LEPROT (19,20,22). Cultured chondrocytes were incubated with 10 5 cpm human 125 I-EGF in the presence or absence of 10 g of unlabeled EGF.…”

“…Previous evidence indicates that fasting-associated increased expression of LEPROT and LEPROTL1 (two transmembrane proteins involved in the regulation of intracellular protein trafficking) reduces GH receptor abundance on the hepatocyte cell membrane (19). Both proteins are also described as endospanin 1 and endospanin 2, respectively (20). Based on this evidence, we hypothesized that the FGF21-dependent reduced GH action in the liver and in the growth plate during chronic undernutrition is mediated by the increased expression of LEPROT and/or LEPROTL1.…”

Endospanin 1 Determines the Balance of Leptin-Regulated Hypothalamic Functions

“…Endo1 is a small protein with 4 transmembrane domains, encoded in humans by the same gene as ObRb via alternative splicing, but without amino acid sequence similarity with the receptor [59, 60]. Besides the genetic link, Endo1 and ObRb display similar expression profiles, in particular in the hypothalamus, and are located in similar intracellular compartments (Golgi, trans-Golgi and late endosomes) [61], suggesting a functional interrelationship. We have shown that Endo1 interacts with ObRb, traps the receptor inside the cell and directs it from endosomes to lysosomes for degradation [61] (Fig.…”