Insulin receptor substrate molecules
Receptor substrate/docking protein
Following insulin binding and receptor autophosphorylation, the next committed step in signal transduction is tyrosine phosphorylation of intracellular proteins. To accomplish this, autophosphorylation of the β subunit mediates noncovalent but stable interactions between the receptor and intracellular substrate proteins, and this positions these molecules for tyrosine phosphorylation by the activated insulin receptor kinase [26 – 28]. Several proteins are rapidly phosphorylated on tyrosine residues by ligand-bound insulin receptors, including six insulin receptor substrate proteins (IRS-1, IRS-2, IRS-3, IRS-4, IRS-5, IRS-6) [29 – 33], Src-homology-collagen proteins (SHC)  and, growth factor receptor bound-2 (Grb2) associated binder-1 (Gab-1) , signal-regulatory protein (SIRP) family members [36,37], the CAP/c-Cbl complex [38,39], an adapter protein with a PH and SH2 domain (APS) , signal transducer and activator of transcription 5B (STAT5B) , and proteins referred to as downstream of kinase (DOK 1-6) [33,42].
There is no known enzymatic activity associated with insulin receptor substrate molecules; however, their primary structure is noteworthy for multiple sites capable of interaction with other proteins. The receptor substrates are characterized by a representative architecture, particularly with respect to three functional domains. These include an N-terminal pleckstrin homology (PH) domain, an intermediate phosphotyrosine binding (PTB) domain, and a carboxy-terminal region of variable length that contains the multiple tyrosine and serine phosphorylation sites [26 – 28]. The PH domain helps position the insulin receptor for coupling with IRS, possibly by binding to charged headgroups of certain phosphatidylinositides in adjacent membrane structures. PTB domains recognize the phosphotyrosine in an amino acid sequence asparagine-proline-any amino acid-phosphotyrosine (NPX pY), and this motif is present in the insulin receptor and other receptors with tyrosine kinase activity such as the IGF-1 receptor. In this manner, the PTB domain enables receptor substrates to directly bind Y972 of the insulin receptor β subunit. All of the receptor substrate molecules have PH domains except SHC, and all have a PTB domain except Gab-1. These domains facilitate binding of the receptor substrates to the phosphorylated insulin receptor, allowing for phosphorylation of tyrosine residues in their COOH-terminal domains.
In the carboxy end of insulin receptor substrate molecules, the phospho tyrosines provide binding sites (pYMM) for other proteins containing Src homology-2 (SH2) domains. This term relates to homology with pp60v-src, the sarc oncogene product and first protein kinase demonstrated to possess activity towards tyrosine residues . Noncovalent attachment of SH2-containing signaling molecules allows receptor substrates to function as docking proteins that appose proteins in the transduction cascade. Receptor substrates thus propagate the insulin signal via the docking, apposition, interaction, and activation of downstream signal molecules, rather than as a consequence of any intrinsic enzymatic activity. This system provides for extensive plasticity and regulation. For example, the multiple receptor substrate molecules exhibit differences in tissue-specific expression and in their capacity to activate various downstream signal pathways. In addition, the receptor substrates can be acted upon by multiple receptor tyrosine kinases, such as IGF-1, interleukin, and integrin receptors, as well as serine kinases (see later), in a manner that allows for modulation of insulin signal and integration of responses to extracellular factors. Thus, these docking molecules provide for divergence, heterogeneity, and regulation of insulin signal transduction processes.
Insulin receptor substrate (IRS) family of docking proteins
IRS-1 and IRS-2 exhibit a wide range of tissue expression, including muscle, fat, liver, and pancreatic islets, although their relative levels can vary in different tissues [27 – 30]. IRS-3 is expressed in adipose tissue, fibroblasts, and liver cells, while IRS-4 has been detected in brain, thymus, and embryonic kidney [31,32]. IRS-5 is ubiquitously expressed but most abundant in kidney and liver. IRS-6 expression is highest in skeletal muscle . Knockout mouse models have highlighted functional differences among the IRS protein family. Genetic ablation of IRS-1 leads to severe growth retardation reflecting a decrease in growth-promoting effects of insulin and other factors which signal through IRS-1 [44,45]. These mice were also insulin resistant but did not develop overt diabetes. On the other hand, ablation of IRS-2 produced insulin resistance, impaired insulin secretion, and overt diabetes in mice that were normal in size . The IRS-2 knockout mice were observed to have decreased β-cell mass and insulin content, indicative of a trophic role for insulin signaling through IRS-2 in β-cell development. Genetic ablation of IRS-2 recapitulates the pathogenesis of T2DM in that diabetes arises only when insulin resistance and insulin secretion are both impaired in these knockout mouse models. IRS-3 knockout mice do not have an obvious phenotype, and IRS-3 has not been detected in the human genome. IRS-4 null mice also appear normal with the exception of reduced fertility. While IRS-5 and IRS-6 undergo insulin-stimulated tyrosine phosphorylation, their relevance to insulin action has been questioned, as both isoforms show very weak affinity for the insulin receptor . Therefore, IRS-1 and IRS-2 are the IRS isoforms most critically important in glucose homeostasis. The consensus that has arisen from these studies is that, while the IRS family members to some extent represent duplicative pathways for insulin signal transduction, IRS-1 functions as the principal IRS in skeletal muscle, and that IRS-2 predominates in liver and β cells where insulin action is required for normal β-cell growth and development.
In addition to PH and PTB domains assuring close apposition to the insulin receptor, IRS-2 (but not other IRSs) contains another region that interacts with the phosphorylated regulatory loop of the insulin receptor kinase, and this region has been designated the kinase regulatory loop binding domain (KRLB) [41,48,49]. Crystal structure analysis of a portion of the KRLB domain indicates that this region acts to restrict tyrosine phosphorylation of IRS-2 . While the KRLB domain could confer some measure of signal specificity, it is unclear whether the pres- ence of the KRLB explains the differences in phenotype between IRS-1 versus IRS-2 knockout mice.
IRS proteins are a key locus for regulation of insulin action. One mechanism occurs at the level of IRS protein expression; for example, IRS-1 and IRS-2 proteins are decreased by hyperinsulinemia . Cell loss of IRS could occur through accelerated protein degradation due to induction of ubiquitin-mediated degradation of IRS-1 and IRS-2 by SOCS proteins , or by inhibition of IRS gene transcription. Regardless of the mechanism, decreased levels of IRS proteins in hyperinsulinemic states, coupled with downregulation of the insulin receptor itself, can contribute to the insulin resistance in diabetes . The function of IRS proteins can also be negatively regulated by serine/threonine kinases and protein tyrosine phosphatases such as PTP1B and SHP2  as described later. In addition, IRS-1 can be posttranslationally modified by either by O-linked N-acetylglucosamine adducts (O-GlcNAc) on serine/threonine residues under hyperglycemic conditions , or by S-nitrosylation as a consequence of nitric oxide generation . These modifications induce the proteasomal degradation of IRS-1 and insulin resistance .
Other receptor substrates
Like IRSs, SHC proteins are phosphorylated in response to insulin, lack any known catalytic activity, and interact with SH-2 domain-containing proteins through their tyrosine phosphorylation sites . A phosphorylation site on SHC binds Grb-2, which can then lead to the activation of the Ras/MAP kinase mitogenic signaling pathway. Therefore, SHC constitutes an additional substrate for the insulin receptor kinase and transduces the insulin signal via its protein docking properties. There are at least three known SHC isoforms (46, 52, and 66 kDa), and all contain an amino terminal phosphotyrosine-binding domain, a central region that is homologous to the α1 chain of collagen, and a carboxy terminal SH-2 domain.
Gab-1 contains only a PH domain at the amino terminus and a domain containing several tyrosine phosphorylation sites, but lacks a PTB domain . In this case, the PH is sufficient for positioning Gab-1 adjacent to the insulin receptor and enabling phosphorylation, although a tight anchor is not possible due to the lack of a PTB domain. Gab-1 is most heavily phosphorylated by the ligand-activated epidermal growth factor receptors, another receptor containing tyrosine kinase activity, and less well by activated insulin receptors.
The DOK family consists of DOK1 through 7 and are similar to IRS family members in domain architecture [33,42,58]. DOK proteins appear to have functional effects in lymphocytes, myeloid cells, muscle cells, and neurons, and can be phosphorylated by both membrane-associated and cytoplasmic tyrosine kinases. DOKs are docking proteins that have been observed to associate with Ras GTPase-activating protein (RasGAP), Nck, c-Abl, and the insulin receptor, and in cultured cells can be involved in cytoskeletal reorganization. DOK1 (p62dok) has been shown to negatively regulate insulin-stimulated activation of AKT , although the role of the remainder of the DOK family proteins in mediating the biologic effects of insulin is not well understood.
Proteins that dock with receptor substrate molecules
SH2-containing proteins, which bind to phosphotyrosine motifs and dock with insulin receptor substrate proteins, include proteins with enzymatic activity such as PI-3 kinase, Fyn (tyrosine kinase), Csk (tyrosine kinase), and SHIP-2 (phosphotyrosine phosphatase), and other adapter proteins such as Grb-2, Crk, APS, and Nck [27,28]. In addition to SH2 domains, the adapter proteins also contain SH3 domains, which bind to proline-rich sequences in other proteins with consensus sequence PXXP. In this way, the adapter proteins associate with receptor substrates via their SH2 domains and bring with them other proteins bound to their SH3 domains. The SH3-bound proteins represent downstream signaling molecules and catalytic subunits, which participate in transduction of the insulin signal. The proteins that dock with receptor substrates, whether binding directly or via adapters, have different enzymatic activities that activate specific downstream molecules as a result of their juxtaposition on receptor substrate docking molecules. In this way, insulin receptor substrates provide the first major point of divergence of insulin signal transduction pathways, leading to activation of the mitogenic (Ras/MAP kinase), and metabolic (PI-3 kinase) pathways. This is illustrated in Figure 12.2.
Ras/MAP kinase pathway: mitogenic signaling
One component of signal divergence emanating from IRS docking proteins is the engagement of the Ras/MAP kinase mitogenic signaling pathway (Figure 12.2). One of the SH-2 domain-containing proteins that docks with IRS is Grb-2, a small cytosolic adapter protein. Grb-2 also contains an SH-3 binding domain which binds proteins via interaction with proline-rich sequences, and one of these proteins is SOS (mammalian homologue of the Drosophila son-of-sevenless protein), a GDP/GTP exchange factor. Following insulin stimulation, Grb-2 is able to bind IRS via its SH-2 domain, and position SOS for activation of the Ras signaling pathway. SOS facilitates GTP activation of membrane-bound Ras, the 21kDa small molecular weight GTPase, which has been demonstrated to play a major role in cell growth and oncogenesis. The GTP-bound form of Ras complexes with and activates Raf-1 kinase, and initiates a cascade leading to sequential phosphorylation and activation of MAP kinase kinase, MAP kinase, and p90RSK. Insulin receptors can also mediate activation of the Ras/MAP kinase pathway through another substrate docking molecule, SHC . Similar to IRSs, SHC activates Ras and the MAP kinase pathway by forming a complex with Grb-2/SOS in response to insulin. Whether activated through IRS or SHC, MAP kinase translocates into the nucleus where it phosphorylates transcription factors mediating the mitogenic and growth promoting effects of insulin . Phosphorylation of nuclear transcription factors modulates their DNA binding properties and ability to regulate gene transcription. For example, p90RSK phosphorylates c-fos, and MAP kinase phosphorylates Elk-1, increasing transcriptional factor activity in both instances. The MAP kinase cascade is also one of the pathways with the potential to stimulate glycogen synthase since p90 S6 kinase is able to activate the glycogen-associated protein phosphatase-1 (PPG-1) which in turn dephosphorylates and activates glycogen synthase. In this way, the MAP kinase pathway has the potential to interact with metabolic signaling pathways (see later). However, the MAP kinase pathway is not necessary for stimulation of glucose transport, and is not viewed as being critically related to the metabolic effects of insulin. For example, inhibition of the pathway in adipocytes using dominant negative forms of Ras  or inhibitors of MAPKK  will block transcriptional effects associated with the MAP kinase pathway, but will not interfere with insulin stimulation of either glucose transport or glycogen synthesis.