Insulin Receptors and Downstream Signaling

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Once in the extracellular environment, insulin can be transported to the major insulin responsive body tissues, also referred to as the triad of skeletal muscle, adipose tissue and the liver, and act as a ligand by binding to its appropriate cell surface receptors. Evidence for a transmembrane receptor specific for the ligand insulin, was first reported by Yip and colleagues (Yip et al., 1978). The IR is now know to be an intrinsic heterotetramer of the form (αβ)₂, belonging to the superfamily of transmembrane receptor tyrosine kinases (Accili et al., 1992; Kahn et al., 1993; Taylor, 1991; White and Kahn, 1994). The α subunits are located extracellular, forming the insulin binding site, whereas the β subunits contain an extracellular portion, a transmembrane domain, and an intracellular part that includes a tyrosine kinase domain (Luo et al., 1999). In contrast to several other tyrosine kinase receptors and independent of ligand binding, the monomeric receptor tyrosine kinases are already covalently linked, forming a dimeric IR complex (Ottensmeyer et al., 2000). Autophosphorylation of the intracellular tyrosine kinase domains is inhibited in the absence of insulin, whereas it is permitted upon insulin binding. Interaction of insulin with the extracellular domain of its receptor, thus results in a conformational change leading to transphosphorylation and activation of the tyrosine kinase domains in the cytoplasmic region  (Saltiel and Kahn, 2001). Certain residues in the tyrosine kinase domains are subsequently recognized by the phosphotyrosine-binding domain of adaptor proteins termed insulin receptor substrates (IRS), which are recruited to the receptor at the cell membrane (see fig. 1). IRS molecules are then phosphorylated by the IR at several tyrosine residues, some of which are recognized by the Src homology 2 (SH2) domain of the p85 regulatory subunit of phosphoinositide 3-kinase (PI3K) (Saltiel and Kahn, 2001). The catalytic subunit of PI3K, p110, then recruits phosphatidylinositol (4,5) biphosphate (PIP2) to the plasma membrane and phosphorylates it at the D3 position of the inositol ring to generate the second messenger phosphatidylinositol (3,4,5) triphosphate (PIP3), which stimulates insulin dependent processes (Vanhaesebroeck et al., 2001). The finding that inhibitors of PI3K or the overexpression of dominant negative mutants of this enzyme, block most of the cellular responses to insulin, including stimulation of glucose transport as well as glycogen and protein synthesis, indicated that PIP3 is a key second messenger in insulin signaling pathways (Lizcano and Alessi, 2002).

An important effector downstream of PIP3 is AKT, also known as protein kinase B (PKB). AKT is a serine/threonine kinase expressed as three isoforms, all containing an N-terminal pleckstrin homology (PH) domain (Brazil and Hemmings, 2001; Lizcano and Alessi, 2002). The PH domain mediates binding to PIP3 and the catalytic domain of AKT containing a threonine residue (Thr305, Thr308, or Thr309 depending on the AKT isoform). Phosphorylation of this residue, in addition to phosphorylation on a second regulatory site positioned on the hydrophobic C-terminal tail (Ser472, Ser473, or Ser474 also depending on the isoform), is required for full activation of AKT (Brazil and Hemmings, 2001). Phosphoinositide-dependent kinase-1 (PDK1), which like AKT has a PH domain and is also recruited by PIP3, phosphorylates AKT at its catalytic domain (Alessi, 2001). It is likely that the co-localization of AKT and PDK1 with PIP3, enables PDK1 to phosphorylate AKT. However, it should be noted that PDK1 is thought to be a constitutively active protein and that it is the substrates of PDK1 that are converted into forms that can be phosphorylated by PDK1. In the case of AKT, the interaction with PIP3 at the plasma membrane converts AKT into a substrate for PDK1. The identity of the protein kinase that phosphorylates AKT at its hydrophobic motif has thus far not been established, although the enzyme has been provisionally termed PDK2. 

Once activated, AKT dissociates from the plasma membrane and phosphorylates numerous substrates in both the nucleus and cytoplasm, which are involved in insulin-mediated processes such as glucose transport, protein and glycogen synthesis, cell proliferation, cell growth, differentiation and survival (Brazil and Hemmings, 2001; Woodgett, 2005). As AKT is thought to have a role in regulation of many biological signaling cascades, it is likely that only a few of the physiological substrates of AKT have thus far been identified. However, one of the most important substrates of AKT is the protein kinase termed glycogen synthase kinase-3 (GSK-3), which is inactivated following its phosphorylation by AKT (see fig. 1) (Cross et al., 1995). GSK-3 is present as two isoforms, i.e. GSK-3α and GSK-3β, and its activities are downregulated by AKT through phosphorylation at Ser21 and Ser9, respectively (Cross et al., 1995; Kaytor and Orr, 2002). One key GSK-3 substrate is glycogen synthase, which catalyzes the final step in glycogen synthesis, the conversion of uridine diphosphate glucose into glycogen (Saltiel and Kahn, 2001). Phosphorylation of glycogen synthase by GSK-3, results in its inhibition. In this case, inactivation of GSK-3 by AKT results in dephosphorylation of glycogen synthase through the action of protein phosphatases and thus results in the activation of glycogen synthesis.

Another major insulin pathway that signals through the IR is associated with the cytoplasmic intermediate protein SHC, which links the IR with other adaptor proteins, i.e. the growth factor receptor-bound protein 2/son-of-sevenless (GRB-2/SOS) complex. Upon interaction of SHC with the IR, its tyrosine residues become phosphorylated by the IR tyrosine kinase activity. When phosphorylated, SHC specifically recruits GRB-2 (which is constitutively bound to SOS) to the IR. SOS is known as a guanylnucleotide exchange protein for the p21 GTP-binding protein RAS. This SHC-mediated recruitment of the GRB-2/SOS complex to the IR, couples the IR to RAS activation (Pawson, 1995; Skolnik et al., 1993), and subsequent activation of mitogen-activated protein kinase (MAPK) cascades (Orban et al., 1999). This specific SHC-mediated insulin signal transduction pathway has been associated with learning and memory function. Several studies consistently report on the involvement of MAPK-activity in various aspects of learning and memory formation in different species (Atkins et al., 1998; Izquierdo et al., 2000; Schafe et al., 1999; Selcher et al., 1999; Zhao et al., 1999). Reports on crosstalk and/or divergence of the insulin signaling pathway, add to the complexity of how insulin regulates many functions, differentially for different tissue types. Therefore it is conceivable that disruption of insulin signaling, perhaps as a result of insulin resistance, can have diverse effects at different levels and at different sites in the body.