One Receptor for Multiple Pathways: Focus on Leptin Signaling

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Leptin is involved in the control of the reproductive system,10 vascular function,11 glucose metabolism,12 immune system and skeleton homeostasis.13,14 Besides its pleiotropic role, leptin is a metabolic hormone that has an important function in the control of energy expenditure, food intake and glucose metabolism. At the hypothalamic level, leptin function as an anorexigenic peptide inducing the expression of specific factors such as cocaine-and-amphetamine- regulated transcript (CART) and proopiomelanocortin (POMC) and inhibits orexigenic neuropeptides such as neuropeptide Y (NPY), agouti-related peptide (AGRP) and orexin. In addition, regarding the straight correlation between circulating leptin levels and adipose tissue mass, leptin levels can be considered as a signal to the body of its energy reserves, and it is likely that the physiological relevance of leptin is based on the fact that low levels signal starvation.15

3.2 Leptin Receptors

Leptin exerts its biological actions through the activation of its cognate receptors, which are encoded by the diabetes (db) gene. These receptors belong to the class I cytokine receptor superfamily, which includes receptors for IL-6, LIF, CNTF, OSM, G-CSF and gp130.16 Db gene alternative splicing gives rise to six receptor isoforms with cytoplasmic domains of di erent length, including one soluble, secreted form (Ob-Re), which lacks the transmembrane region, and four short forms (Ob-Ra, Ob-Rc, Ob-Rd and Ob-Rf), which have slight di erences in the length of their cytoplasmic domains and share the extracellular domain, the transmembrane domain and a small intracellular fragment. Finally, only the one long-functional isoform (Ob-Rb), which has the full intracellular domain with the typical signaling elements of cytokine receptors17,18 is able to transduce the signal to the nucleus. Despite the fact that Ob-Ra is the most expressed form; it does not exhibit the ability to transduce properly the leptin signal. In contrast, the distribution of Ob-Rb, the functional form, correlates with leptin in proposed target tissues such as hypothalamus and peripheral tissues, like WAT.19 However, it must be noted that, even though the short Ob-R forms (Ob-Ra and Ob-Rc) are devoid of full signaling, it has been described that they participate in leptin uptake and leptin availability for Ob-Rb.20,21

3.3 Leptin Receptor Signaling

3.3.1The JAK2-STATs Routes

Leptin receptor (Ob-Rb), as a member of the class I cytokine receptor superfamily, does not have intrinsic tyrosine kinase activity. Due to this fact, it must recruit cytoplasmic kinases like the Janus kinase family JAK2 to achieve leptin signaling.22 The activation of JAK2 kinase by the leptin receptor is supported by the constitutive interaction between JAK2 kinase and the leptin receptor.23 In this situation, the ability of this receptor to form aggregates (homodimers),

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in a ligand-independent way, facilitates the autophosphorylation of JAK2, eliciting leptin signaling.24,25 In this sense, it has been shown that two JAK2

molecules are needed for the signaling pathway activation.23 Once JAK2 is activated, it phosphorylates three tyrosine residues (Tyr985, Tyr1077, Tyr1138) of the murine leptin receptor.24,26 These tyrosine residues are highly conserved, which means they are very important for leptin signaling. Accordingly, it has been determined that these conserved tyrosine residues behave as docking sites for proteins with SRC homology 2 domains, such as STATs (Signal Transducers and Activators of Transcription) proteins, which have high a nity for phosphotyrosine residues.26,27

Each tyrosine phophorylation site is involved with the recruitment of different signaling proteins.27 Among these proteins there are STATs. These kinds of proteins are the most known targets of JAK activation. After binding to the phosphotyrosine residues of the leptin receptor, these signaling molecules are also phosphorylated by receptor associated JAK kinases (RAJK), and are involved in dimerization and translocation to the nucleus, where they function as transcription factors.27

One of the major STATs proteins involved in leptin signaling is STAT3. Activation of this STAT protein, after leptin binding to its receptor, has been

found in multiple cell types such as hypothalamic neurons, adipocytes, immune cells and pancreatic cells.28–31 Ob-Rb has a consensus binding site region for

STAT3. This region, also named Box3, has the structure Tyr-X-X-Gln from position 1138 to 1141 and is essential for leptin signaling.26 In fact, mutational analysis of box3 around Tyr1138 confirmed that this motif is necessary for STAT3 signaling in addition to other STATs.26 In this sense, it has been described that leptin has the ability to signal through the recruitment and activation of

other STATs di erent from STAT3. Among these STATs are STAT1, STAT5 and STAT6.26,32 The exact role of these proteins in leptin signaling is still unclear.

However, it is known that several residues of the leptin receptor are necessary for its recruitment, such as Met 1139 and Gln 1141.26 So, leptin behaves as other members of the class I cytokine receptor superfamily such as IL-6,33 inducing the activation of STAT1 and STAT3 through the above-mentioned conserved motif box3. According to this, it has been described that STAT1 signaling is involved in some leptin actions such as immune competence against microbial aggression.34 Likewise, recently, it has been determined that STAT1 signaling is involved in leptin inhibition of peroxisome proliferator activated receptor gamma (PPARg) expression.35 Moreover, STAT1 is also involved in leptin inhibition of adipocyte di erentiation and lipid accumulation in murine primary adipocytes.35 On the other hand, it must be considered that interactions among di erent STATs proteins may also be at play since, in other members of the of the class I cytokine receptor superfamily, STAT1 signaling is possibly blunted by STAT3 activation.36 Strengthening this idea is the fact that ASKO mice, a murine strain with an adipocyte specific disruption of the STAT3 gene, have a strong STAT1 signaling in comparison to WT littermates.37

In addition to STAT3 and STAT1, leptin signaling, through the activation of the leptin long form receptor, also involves other STATs proteins. It has been

One Receptor for Multiple Pathways: Focus on Leptin Signaling

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Figure 3.1 Schematic representation of leptin receptor signal transduction pathways. Upon leptin binding, leptin receptor initiates multiple intracellular signaling routes that in turn lead to the activation of ERK pathway, PI3/ AKT pathway, AMPkinase pathway and STAT transcription factors.

described that the leptin receptor is able to recruit and activate STAT5 in two di erent phosphotyrosine residues, namely Tyr1077 and Tyr1138. As already mentioned above, it is noteworthy that the 1138 tyrosine residue is also considered part of the STAT3 recruiting mechanism.26,38 Although the functional relevance of leptin-induced STAT5 activation still has to be determined, several

studies point to a role for STAT5 in leptin action at the hypothalamic and pancreatic levels.38–40 It is important to note that, as observed for STAT1

signaling, STAT5 signaling is also under the control of STAT3 activation. Indeed, Gong et al. described the existence of a Tyr1138/STAT3-mediated

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feedback inhibition that attenuates STAT5-dependent transcription during chronic receptor activation.38

3.3.2ERK1/2

Another important pathway elicited by leptin receptor activation is the ERK

signaling pathway. Leptin has the ability to activate the extracellular signal regulated kinase (ERK) pathway18,27,41 in multiple cellular systems. According

to that, activation of the ERK pathway also has been reported for other members of the cytokine class I family of receptors.42 The ability of the leptin receptor to activate the ERK pathway is mediated by the phosphotyrosine residue Tyr985 generated by JAK2 activity. This residue behaves as a binding site for an SH2 domain containing protein, namely SHP-2.43 This interaction, in turn, induces firstly the activation of this protein and next the recruitment of growth factor receptor-bound protein 2 (GRB2).27 Once this adaptor protein is recruited, the signaling pathway elicited by leptin receptor activation leads to the activation of ERK.

Bjorbaek et al. have demonstrated that two di erent pathways are critical for leptin-receptor-mediated ERK activation. In these studies, both dependent and independent Tyr985-mediated mechanisms for ERK activation have been demonstrated. Moreover, it has also been described that the SHP-2 protein is essential for both pathways.44 According to this, it has been suggested that leptin also has the ability to induce ERK activation in a Tyr985-independent manner through a direct interaction between GRB2 and JAK2.27

As mentioned above, several activities of the leptin receptor are common among members of the cytokine class I family of receptors. In this sense, it has been reported that SHC, GRB2 and SHP-2 proteins directly bound to the murine granulocyte colony-stimulating factor receptor suggesting multiple routes for ERK activation.45 According to this, Gualillo et al. have reported that after leptin receptor activation, SHC proteins were phosphorylated. This process induces the recruitment of GRB2 and the activation of the ERK pathway.41

After leptin receptor stimulation, the functional role of ERK activation has been related with the regulation of cell survival and to the control of linear growth in several di erent cell types.28 According to these roles, very recently it has been reported that leptin protects neurons against glucose-oxygen-serum-deprivation (GOSD) injury through ERK activation.46 Moreover, leptin-receptor-mediated activation of ERK also inhibited the apoptosis of trophoblastic cells.47 In addition to this protective role, ERK activation has been involved in leptininduced proliferation of hepatic, endometrial and renal cells.48–50

However, despite the above-described functions for leptin-mediated ERK activation, other functions have been described. Namely, hypothalamic ERK activation by leptin mediates the anorectic and thermogenic e ects of leptin.51 Another function mediated by leptin-induced ERK activation (through the phosphotyrosine residue Tyr985) is the phosphorylation of the ribosomal protein S6 kinase (RSK), which in turn leads to modulation of cap-dependent translation.38