Editorial: Insulin-Like Growth Factor I Receptor Signaling—Overlapping or Redundant Pathways?

The insulin-like growth factor (IGF) family of ligands and receptors are members of a highly conserved growing family of insulin-related peptides and receptors that are ubiquitously expressed. The IGF family (IGF-I, IGF-II) is distinct from insulin in the existence of a large family of high-affinity binding proteins (IGFBPs). The IGFs play important roles in numerous physiological processes. These processes range from normal growth and development during the early stages of embryogenesis to the regulation of specific functions for various tissues and organs in later stages of development. The IGFs are essential for an array of diverse processes, from progression of the cell cycle, which triggers cellular proliferation, to regulation of programmed cell death (apoptosis). These growth factors also induce cellular differentiation and stimulate certain enzymatic functions in specialized tissues. In the reproductive system, IGFs augment steroidogenesis and stimulate proliferation and differentiation of bone cells, muscle cells, and cells derived from the hematopoietic and lymphoid systems, to mention just a few examples. The total concentrations of IGF-I in the circulation are closely correlated with longitudinal growth. This and other evidence suggests that during both normal and pathophysiological situations, such as dwarfism and acromegaly, circulating IGF-I regulates body growth. In addition, locally produced IGF-I mediates critical autocrine/paracrine effects on tissues. Whatever the source of the IGFs, their biological actions are predominantly mediated by the IGF-I receptor (type 1) receptor (1). While the closely related insulin receptor is capable of binding the IGFs, this interaction is of lower affinity than of insulin for its own receptor. However, under certain circumstances, the insulin receptor may also mediate some of the biological actions of the IGFs. On the other hand, the IGF-II/mannose-6-phosphate (type 2) receptor does not appear to regulate any significant signaling cascades. Like the insulin receptor, the insulin-like growth factor I receptor (IGF-IR), is a member of the receptor tyrosine kinase family of growth factor receptors. The IGF-IR is expressed at the cell surface as a tetramer, comprised of two α and two β subunits. The α subunits are primarily localized extracellularly and mediate ligand binding, whereas the two β subunits are primarily intracellular and possess intrinsic tyrosine kinase activity. The IGF-IR binds IGF-I and IGF-II with high affinity, and insulin at a considerably lower affinity. Ligand binding to the extracellular receptor triggers autophosphorylation of the β subunits and stimulates the tyrosine kinase activity. This sequence of events involves a conformational change in the catalytic loop domain of the tyrosine kinase region, binding of ATP to residue lys1003, and phosphorylation of residues tyr1131, tyr1135, and tyr1136. Eachβ subunit then transphosphorylates the other, leading to phosphorylation of a number of other tyrosines including, but not limited to, tyr950 in the juxtamembrane region, tyr1250, tyr1251 and tyr1316 in the carboxyl-terminal domain of theβ subunit. Substitution of phenylalanine for tyrosine in each of these residues has resulted in a loss of function (2–7). Tyrosine phosphorylation of the IGF-I receptor has two distinct, but related, outcomes. Firstly, the tyrosine kinase activity of receptor is enhanced, and secondly, the phosphorylated tyrosine residues provide docking sites for various proteins that mediate the signaling cascades emanating from the IGF-I receptor. Previous studies have indicated that the juxtamembrane region of the cytoplasmic domain of the β-subunit binds both Shc and the various isoforms of the IRS family of proteins. Binding of IRS proteins to the IGF-I receptor occurs via the NPXpY motif, which includes tyr950 (8, 9). Both IRS proteins and Shc proteins contain SH2 domains that bind to the activated receptor. After binding to the receptor, these proteins become phosphorylated on tyrosine residues, presumably by the IGF-I receptor. Grb2 (growth factor receptor-bound protein 2), an adapter protein, binds both Shc and IRS via its SH2 domain and via its SH3 domain to mSOS (mammalian Son of Sevenless) (10). mSos is a guanine nucleotide-exchange protein that loads GTP onto the small G protein Ras, and thereby activates the Ras/Raf/MAP kinase pathway. Tyrosine-phosphorylated IRS molecules bind the regulatory subunit (p85) of phosphoinositide 3′-kinase (PI3′K) via its SH2 domain and p85 binds the catalytic subunit of PI3′K via its SH3 domain. While other substrates are currently under investigation, these two major pathways have been identified as playing important roles in IGF-IR-induced cellular proliferation and the inhibition of apoptosis. Traditionally, the Ras/Raf/MAP kinase pathway was thought to primarily mediate the cell proliferative response to growth factors such as the IGFs, whereas the PI3′kinase pathway, which activates AKT/PKB, was primarily implicated in mediating the antiapoptotic effects of the IGFs (11–13). However, recent studies have demonstrated a role for both pathways in mediating both responses. Naturally occurring genetic mutations in the insulin receptor have been identified in patients suffering from severe insulin resistance (14). In contrast, no such mutations have been reported in the case of the IGF-IR. Thus, a number of investigators have resorted to creating amino acid substitutions in the IGF-IR to investigate the importance of the various domains in the function of the receptor. These studies have generally proven successful and have identified specific regions of the IGF-IR that play critical roles in cell proliferation or inhibition of apoptosis. Substitutions in the tyrosine kinase domain of the ATP-binding site (lys1003) results in a kinase-deficient receptor. Interestingly, this mutant functions in a dominant-negative manner, whereas mutations of the triple tyrosine motif (tyr1131, tyr1135 and tyr1136) result in a kinase-deficient, but not dominant-negative receptor (4, 5). Both mutations result in total abrogation of IGF-IR function. Substitution of phenylalanine for tyrosine at tyr950 resulted in the loss of receptor binding to Shc and IRS molecules and interfered with IGF-I-induced mitogenic signaling and inhibition of apoptosis. While the juxtamembrane region appeared to be critical for the activation of the MAP kinase and PI3′K pathways, it soon became apparent that the C-terminal domain was also important in the function of the IGF-IR. This was determined by C-terminal deletions, which severely affected the function of the receptor (15). More specific amino acid substitutions in the C-terminus were also instructive. That is, substitution at both tyr1250 and tyr1251 inhibited cellular proliferation, whereas substitution of the distal tyrosine (tyr 1316) did not (2). Thus, it is apparent from these and other studies that the receptor has specific domains involved in its function and that these phosphotyrosines interact with both known and currently unidentified substrates. In this issue, Baserga and colleagues present studies that have extended these findings in a very elegant manner (16). Using cells that are do not express IRS-1 or IRS-2 (32D cells originally developed by Dr. Jackie Pierce, NIH, Bethesda, MD) they have dissected the pathways involved in cell survival. Following cytokine withdrawal, 32D cells undergo apoptosis, which can be prevented by IGFs activating the IGF-IR. In the present study, the investigators have established that in the absence IRS molecules (at least IRS-1 and IRS-2 because IRS-3 and IRS-4 levels were determined), IGF-IR activation of the MAP kinase pathway persisted. This particular finding is not unexpected to the casual reader because it has been well established that the IGF-induced activation of MAP kinase pathway can be mediated through Shc. What may be less obvious, is that MAP kinase activation prevents apoptosis. While this has been previously established in another cell line (17), the present study confirms that this is likely not to occur through IRS molecules, which, in addition to activating MAP kinase, also strongly activate the PI3′K/AKT antiapoptotic pathway. While these findings are strongly confirmatory of previous data, they were extended and revealed even more exciting results. A quartet of serine quartet residues, located at 1280–1283 in the C-terminal domain of the IGF-IR, is not present in the insulin receptor. This serine quartet, along with tyr950, was shown to be critical for maximal activation of MAP kinase by IGF-I, as mutation of these two regions together, but not separately, abrogated activation of MAP kinase by IGF-I. While tyr950 has been previously shown to interact with Shc, the molecular mechanisms that serines 1280–1283 are involved in are not yet defined. 14–3-3 proteins are obvious candidates because certain isoforms activate Raf, and recently the β isoform of 14–3-3 has been shown to bind to this region (18–20). It is important to consider the usual caveats when interpreting the results of these studies. The response of cells to various stimuli is frequently cell-type specific and is also dependent on the specific cellular context, i.e. what other stimuli are affecting the cell at the same time. In addition, overexpression of proteins in cell lines may alter certain physiological functions. Nevertheless, these and other studies are rapidly enhancing our understanding of the structure-function relationship of the IGF-IR. Furthermore, these findings will facilitate the identification of agents that can either enhance IGF-IR function (and thereby inhibit apoptosis and stimulate cellular proliferation) or inhibit IGF-IR function, which may be helpful in adjunct therapy for some common malignancies.