IRR is a member of the insulin receptor (IR) family that does not have any known agonist of a peptide nature but can be activated by mildly alkaline medium and was thus proposed to function as an extracellular pH sensor. IRR activation by alkali is defined by its N-terminal extracellular region. To reveal key structural elements involved in alkali sensing, we developed an in vitro method to quantify activity of IRR and its mutants. Replacing the IRR L1C domains (residues 1–333) or L2 domain (residues 334–462) or both with the homologous fragments of IR reduced the receptor activity to 35, 64, and 7% percent, respectively. Within L1C domains, five amino acid residues (Leu-135, Gly-188, Arg-244, and vicinal His-318 and Lys-319) were identified as IRR-specific by species conservation analysis of the IR family. These residues are exposed and located in junctions between secondary structure folds. The quintuple mutation of these residues to alanine had the same negative effect as the entire L1C domain replacement, whereas none of the single mutations was as effective. Separate mutations of these five residues and of L2 produced partial negative effects that were additive. The pH dependence of cell-expressed mutants (L1C and L2 swap, L2 plus triple LGR mutation, and L2 plus quintuple LGRHK mutation) was shifted toward alkalinity and, in contrast with IRR, did not show significant positive cooperativity. Our data suggest that IRR activation is not based on a single residue deprotonation in the IRR ectodomain but rather involves synergistic conformational changes at multiple points.

The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane. We showed that with excitation at 445 nm, the fluorescence lifetime of both SypHer3s and SypHerExtra strongly depend on pH. Using FLIM microscopy in live eukaryotic cells, we demonstrated that SypHerExtra can be successfully used to determine extracellular pH, while SypHer3s can be applied to measure intracellular pH. Thus, these two sensors are suitable for quantitative measurements using the FLIM method, to determine intracellular and extracellular pH in a range from pH 7.5 to 9.5 in different biological systems.