Mapping of alkali-sensing sites of the insulin receptor-related receptor. The role of L2 and fibronectin domains.

Insulin receptor-related receptor (IRR) is a member of the insulin receptor (IR) family that works as an extracellular alkali sensor with positive cooperativity. The pH sensing property of IRR is defined by its extracellular region and involves multiple domains. We have previously demonstrated the primary role of L1C domains and identified potentially important amino acid residues within these domains. In this study, we addressed the roles of L2 and FnIII domains. Within the L2 domain, five amino acid residues (M406, V407, D408, P436 and V437) were identified as IRR-specific by performing a species conservation analysis of the IR family. Single-point mutations of these five residues to alanine produced either little or no negative effect on IRR pH-sensing activity. However, the triple mutation of M406, V407 and D408 (MVD) showed a strong negative effect, with a 4 fold decrease in IRR activity as estimated by in vitro autophosphorylation assay of solubilized receptors. The analysis of this mutant in intact cells revealed the absence of positive cooperativity. Unexpectedly, the double mutation of vicinal P436 and V437 (PV) exhibited a significant positive effect in the in vitro assay and partial positive cooperativity in the whole-cell assay. The role of FnIII domains was addressed by analyzing chimeras of IRR and IR. When the IRR FnIII domains were swapped with those of IR in different combinations, the activity was significantly reduced and positive cooperativity eliminated. However, two mutants with the targeted C-terminal part of IRR alpha subunit that lies within FnIII-2 domain and have been shown to be important for insulin binding by IR, appeared to be as active as wild-type IRR. On the basis of available data, we propose that IRR activation involves two separate centers of pH-dependent rearrangements that act synergistically to induce a major conformational change in the IRR molecule, resulting in internal kinase domains rapprochement and autophosphorylation.