Abstract

Receptor tyrosine kinases (RTKs) are critical mediators of cellular signaling, and their activity is often tightly regulated by the extracellular environment, including pH. The insulin receptor-related receptor (IRR), an orphan RTK primarily expressed in the kidney and pancreas, is unique among its family members for its pronounced pH sensitivity, which is hypothesized to play a role in its physiological function. This study aimed to precisely identify the specific amino acid residues within the IRR ectodomain responsible for mediating this pH-dependent conformational change and subsequent kinase activation. Using site-directed mutagenesis, followed by biochemical assays including ligand binding, autophosphorylation kinetics, and cell-based signaling reporter systems, we systematically screened candidate histidine and glutamic acid residues. Our findings reveal that a cluster of three histidine residues (His-X-His-Y-His) in the juxtamembrane domain are essential for the pH-sensing mechanism. Mutation of these residues to alanine significantly shifted the pKa of IRR activation, rendering the receptor constitutively active or insensitive to physiological pH fluctuations. These results provide a molecular basis for the pH-dependent regulation of IRR, offering crucial insights into the mechanisms governing RTK function in response to environmental cues and highlighting a novel regulatory paradigm for this important class of membrane proteins.