Subcellular Localization and Internalization of the Four Human Leptin Receptor Isoforms

Abstract

There are four known isoforms of the human leptin receptor (HLR) with different C-terminal cytoplasmic domains (designated by the number of unique C-terminal amino acids). In cells expressing HLR-5, -15, or -274, 15-25% of the leptin binding sites were located at the plasma membrane. In contrast, in cells expressing HLR-67, only 5% of the total binding sites were at the plasma membrane. Immunofluorescent microscopy showed that all four isoforms partially co-localized with calnexin and ␤-COP, markers of the endoplasmic reticulum and the Golgi, respectively. All isoforms were also detected in an unidentified punctate compartment. All isoforms were internalized via clathrin-mediated endocytosis, but at different rates. After 20 min at 37°C, 45% of a bound cohort of labeled ligand had been internalized by HLR-15, 30% by HLR-67, 25% by HLR-274, and 15% by HLR-5. Degradation of internalized leptin occurred in lysosomes. Overnight exposure to leptin down-regulated all isoforms, but to a variable extent. HLR-274 displayed the greatest down-regulation and also appeared to reach lysosomes more quickly than the other isoforms. The faster degradation of HLR-274 may help to terminate leptin signaling.Leptin is a peptide secreted primarily by adipose cells that regulates appetite, energy metabolism, and neuroendocrine function. Leptin acts both centrally, presumably in the hypothalamus, and directly on peripheral tissues (1-3). Leptin binding activates its receptor, a member of the cytokine receptor superfamily, which includes receptors for interleukins, prolactin, growth hormone, and erythropoietin (4, 5). As a result of differential mRNA splicing, there are several isoforms of the leptin receptor with different lengths and C-terminal sequences. The regions that are identical in all the receptor isoforms include the extracellular ligand binding domain, the transmembrane domain, and the first 29 amino acids in the cytoplasmic domain. (4, 6 -11).Some cytokine receptors (e.g. receptors for growth hormone, erythropoietin, and prolactin) form homodimers when activated by ligand, while others form hetero-oligomers (12)(13)(14). Recent studies have shown that leptin receptors form homodimers, both in the presence and absence of ligand (15, 16). Each leptin receptor binds one molecule of leptin, resulting in a tetrameric complex composed of two receptors and two leptin molecules. However, activation of the receptor is thought to result from a ligand induced conformational change rather than dimerization of the receptor (15, 17). All the leptin receptor isoforms contain a "box 1" Janus kinase binding site in the cytoplasmic domain. The longest form also contains a "box 2" motif and putative STAT 1 binding sites (5, 10, 11, 18) and thus only the long form is able to activate STAT proteins (18 -20).Receptor-mediated endocytosis is a well characterized mechanism for selectively transporting nutrients, hormones, and growth factors into cells. Often receptors are concentrated in clathrin-coated pits and then internalized in clathrin-coate...

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

“…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.…”

Leptin and cancer

“…In human tissues, at least four isoforms of ObR with different COOH-terminal cytoplasmic domains have been described (Barr et al, 1999). The full (long) form of ObR (ObRl) is 1,165 amino acids long (Mr 150,000) and contains the extracellular, transmembrane, and intracellular domain (Fig.…”

“…Ultimately, induction of ObRl can activate several genes involved in cell proliferation, including c-fos, c-jun, junB, egr-1, and socs3, and upregulate the expression angiogenic factors, Fig. 1 (Barr et al, 1999). SRL is a soluble leptin receptor generated by MMPs-mediated shedding of ObRl or ObRs ectodomains.…”