Low Levels of Expression of Leptin Receptor at the Cell Surface Result from Constitutive Endocytosis and Intracellular Retention in the Biosynthetic Pathway

Abstract

The leptin receptor is mainly localized in intracellular compartments in target tissues. To study the mechanisms leading to this intracellular localization, two main isoforms of leptin receptors, OB-Ra and OB-Rb, were expressed in HeLa cells. Both isoforms were localized at steady state in the trans-Golgi network, in endosomes, and to a lesser extent, at the cell surface. They turned over with a half-life of less than 2 h. Both isoforms of leptin receptors were constitutively endocytosed in a ligandindependent manner and degraded in lysosomes with no evidence of recycling to the cell surface or to the transGolgi network. The endocytosis was inhibited by the deletion of the cytoplasmic domain. Newly synthesized leptin receptors were partially retained in the Golgi complex or in a post-Golgi intracellular compartment. The transmembrane domain was found to be important for this intracellular retention in the biosynthetic pathway, whereas the cytoplasmic domain was not involved. The data suggest that the low levels of expression of leptin receptors at the cell surface results from partial retention in the biosynthetic pathway, coupled to constitutive removal from the plasma membrane via ligand-independent, constitutive endocytosis.Leptin is a polypeptide hormone secreted by adipocytes, which plays a major role in the regulation of food intake, energy expenditure, and neuroendocrine functions (1, 2). Leptin action is mediated by receptors that are located in the hypothalamus and in many peripheral tissues. Leptin receptors (OB-R) 1 are type I membrane glycoproteins structurally related to class I cytokine receptors (3). Different OB-R isoforms have been described, including OB-Ra and OB-Rb. OB-Rb is often referred to as the long isoform of the leptin receptor. OB-Ra, a short isoform, is the major form of leptin receptor found in most peripheral tissues. Both OB-Ra and OB-Rb are co-expressed in a large number of tissues.All known OB-R isoforms originate from a single gene by alternative splicing of the transcript and only differ in their C termini. OB-Ra and OB-Rb share identical N-terminal leptin binding domains, single spanning transmembrane (TM) domains, and the 29 proximal residues of their cytoplasmic domains. They diverge in the C-terminal part of their cytoplasmic domains. OB-Ra has a unique 5-residue long C-terminal extension, which is changed in OB-Rb for a 273-residue long extension (3). OB-Rb has been shown to activate various signal transduction pathways. The best characterized leptin-induced transduction pathway triggered by OB-Rb is Janus tyrosine kinase 2 (JAK2)-mediated activation of signal transducer and activator of transcription 3 (STAT3) (4, 5). OB-Rb was also found to activate the phosphoinositide 3-kinase (6), mitogen-activated protein kinase (7), and 5Ј-AMP-activated protein kinase (8) pathways in response to leptin. In contrast, the function of OB-Ra remains undefined. It has been shown that OB-Ra could bind to and activate JAK2 in vitro (9, 10), but there is no evidence that OB-Ra actually has...

Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity

“…Viral stocks were generated, amplified, and tittered by the TCID50 method in N52.E36 cells. Adenoviruses expressing the HA-tagged OB-Ra or OB-Rb have been described (21).…”

“…Little is known about the intracellular trafficking of OB-R (19,21,26,27). We recently showed that OB-R internalizes via a clathrin-mediated pathway and is transported to lysosomes where it is degraded.…”

“…We recently showed that OB-R internalizes via a clathrin-mediated pathway and is transported to lysosomes where it is degraded. Most OB-Rs (endogenously expressed or transfected) are localized in intracellular membranes (16)(17)(18)(19)(20)(21)28). Importantly, these receptors are fully functional in terms of ligand binding (19,20).…”

“…Possible mechanisms underlying this pathological state, termed leptin resistance, are impaired leptin bioavailability and transport across the blood-brain barrier, up-regulation of negative feed-back regulators of OB-R signaling, and defects in OB-R trafficking and signaling (13)(14)(15). Importantly, at steady state, most OB-R (endogenously expressed and transfected) are in intracellular membranes (16)(17)(18)(19)(20)(21) and are fully functional in terms of ligand binding (19,20). However, they are unable to participate in the functional response, as leptin does not penetrate the cell.…”

Reciprocal cross-regulation between RNF41 and USP8 controls cytokine receptor sorting and processing

“…2A,C, lane 2 versus 1). The LR constitutively internalizes in a leptin-independent manner (Belouzard et al, 2004). After internalization, it was previously reported that the LR does not appear to recycle, but is transported via Rab5-positive early endosomes and Rab7-positive late endosomes to the lysosomes, where LR degradation occurs (Belouzard and Rouillé, 2006).…”