Interaction between the Insulin Receptor and Its Downstream Effectors

Abstract

A structural analysis has been carried out to determine which part of the intracellular domain of the insulin receptor (IR) ␤ subunit is involved in direct interaction with the receptor substrates IRS-1 and Shc. Toward this end, the juxtamembrane (JM) domain (amino acids 943-984) and the carboxyl-terminal (CT) region (amino acids 1245-1331) of IR were expressed in bacteria as (His) 6 -fusion peptides, and their interaction with IRS-1 and Shc was studied. We could demonstrate that the CT region of IR was sufficient to bind Shc, although significant, but much lower binding of Shc to the JM region could be detected as well. Furthermore, in vitro Tyr phosphorylation of the CT region potentiated its interactions with Shc 2-fold. In contrast, the JM region, but not the CT domain of the IR, was sufficient to mediate interactions between the IR and IRS-1. These interactions did not involve the pleckstrin homology (PH) region of IRS-1, since an IRS-1 mutant, in which four "blocks" of the PH domain (Pro 5 -Pro 65 ) were deleted, interacted with the JM region of IR with the same efficiency as native IRS-1. These results suggest that the IR interacts with its downstream effectors through distinct receptor regions, and that autophosphorylation of Tyr residues located at the CT domain of the IR can modulate these interactions. The insulin receptor (IR)1 is an heterotetrameric transmembrane glycoprotein composed of two extracellular ␣ subunits and two transmembrane ␤ subunits linked by disulfide bonds. The ␣ subunits contain the insulin-binding domain, while the transmembrane ␤ subunits function as a tyrosine-specific protein kinase that undergoes autophosphorylation following insulin binding (for reviews, see Refs. 1 and 2). Autophosphorylation activates the insulin receptor kinase (3) and enables it to phosphorylate endogenous proteins, including insulin receptor substrate-1 (IRS-1) (4), IRS-2 (5), and Shc (6).Several structural regions have been defined within the intracellular part of the ␤ subunit. These are the juxtamembrane (JM) region, the kinase region, and the carboxyl-terminal (CT) region (7,8 ), which resides in an LX 4 NPXYXSXSD motif (numbering of IR amino acids is according to Ullrich et al. (7)). Replacement of Tyr 960 with Phe or Ala impairs receptor signal transmission and abolishes both the metabolic and growth-promoting effects of insulin, even though autophosphorylation in other regions is normal and the kinase is fully active in vitro (9, 10). This appears to be due to an inability of the mutant receptors to mediate the phosphorylation of endogenous receptor substrates, including IRS-1. Indeed, overexpression of IRS-1 can rescue certain biological effects in cells overexpressing the mutated receptor (1). Similarly, the IGF-1 and the IL-4 receptors, which contain LX 4 NPXYXS motifs, also phosphorylate IRS-1 (11, 12). These findings strongly suggest that the JM region of IR is involved in the interactions with IRS-1.The CT region (Leu 1245 -Ser 1343) possesses only limited (44%) homology with the related IG...

A Molecular Basis for Insulin Resistance

“…In the present study we undertook to address this issue by eliminating the effects of Ser/Thr phosphorylation of IR, focusing only on changes in Ser(P)/Thr(P) content of IRS-1 and IRS-2. We took advantage of our previous findings that the isolated juxtamembrane (JM) domain of IR (amino acids 943-984),but not its carboxyl-terminal (CT) region (amino acids 1245-1331),is sufficient to mediate interactions between IR and IRS-1 (18). Here we present evidence that similar to IRS-1, IRS-2 also interacts with the JM but not the CT region of IR.…”

“…Peptide Expression and Purification-(His) 6 -tagged fusion peptides corresponding to 41 amino acids (amino acids 943-984) of the juxtamembrane region of IR ((His) 6 -JM) or 86 amino acids (amino acids 1245-1331) of the carboxyl-terminal region of IR ((His) 6 -CT) were generated in bacteria and purified over ProBond Ni 2ϩ beads (Invitrogen) as described (18). Synthetic peptides with the following sequence were used for binding-inhibition experiments: P 951 pep (PLYASSNPEYLSAS-NH 2 ) and S 1315 pep (SYEEHIPYTHMNG-NH 2 ).…”

“…The beads were washed four times with buffer A containing 200 mM imidazole and 0.1% Nonidet P-40, washed two times with buffer A, and boiled in 60 l of Laemmli "sample buffer" (22). Samples were resolved by means of SDS-PAGE and subjected to Western blotting with the appropriate antibodies as described (18).…”

“…Aliquots (300 g, ϳ100 l) were incubated with 5 l (1000 units) of alkaline-phosphatase for 1 h at 37°C. Following incubation, samples were subjected to precipitation using immobilized JM peptide (see above), boiled in 60 l of Laemmli sample buffer (22), resolved by means of SDS-PAGE, and subjected to Western blotting with IRS-1 or IRS-2 antibodies (18).…”

Molecular Mechanisms of Insulin Resistance: Serine Phosphorylation of Insulin Receptor Substrate-1 and Increased Expression of p85α

“…Once the tyrosine kinase of insulin receptors is activated, it promotes autophosphorylation of the ␤ subunit itself, where phosphorylation of three tyrosine residues (Tyr-1158, Tyr-1162, and Tyr-1163) is required for amplification of the kinase activity (12,13). Activation of the tyrosine kinase of the insulin receptor also leads to a rapid phosphorylation of the so-called "docking proteins," such as insulin receptor substrate (IRS)-1, -2, -3, and -4, and several Shc proteins (52-, 46-, and 64-kDa isoforms) (14,15) that, in turn, attract multiple intracellular signaling intermediates.…”