Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

<b>Background:</b> Intracellular delivery of high-molecular-weight proteins is limited by the cell membrane. Cell-penetrating peptides (CPPs) offer a potential solution, but effective cytosolic delivery remains hindered by endosomal sequestration. Pas2r12, a CPP-derived peptide, facilitates cytosolic delivery of proteins including immunoglobulin G. Because Pas2r12 internalization occurs via caveolae-dependent endocytosis, we hypothesized that cell-surface receptors contribute to uptake. <b>Methods:</b> HEK293 cells were treated with Pas2r12 alone or complexed with enhanced green fluorescent protein (EGFP). Phosphorylation of insulin receptor (INSR), insulin-like growth factor 1 receptor (IGF1R), and extracellular signal-regulated kinase 1/2 (ERK1/2) was analyzed by Western blot. Linsitinib was used to inhibit INSR/IGF1R kinase activity. Cytosolic delivery was assessed by confocal microscopy, and receptor involvement was evaluated using siRNA-mediated knockdown and receptor overexpression. <b>Results:</b> Pas2r12 alone transiently increased INSR/IGF1R phosphorylation at 2 min (6.6-fold), which was suppressed by linsitinib (1.3-fold), and strongly increased ERK1/2 phosphorylation (6.2-fold), which was not inhibited by linsitinib. Pas2r12-EGFP did not induce detectable INSR/IGF1R phosphorylation in parental cells but increased ERK1/2 phosphorylation (3.4-fold). Linsitinib markedly reduced cytosolic EGFP delivery to 16% of control. INSR knockdown decreased delivery to 13-16%, and IGF1R knockdown to 19-65%. In INSR-overexpressing lines, Pas2r12-EGFP induced INSR/IGF1R phosphorylation (6.0-fold) and enhanced delivery (230-270%). In IGF1R-overexpressing lines, Pas2r12-EGFP did not induce phosphorylation, and delivery decreased to 60-69%. <b>Conclusions:</b> Pas2r12-mediated cytosolic delivery involves both INSR and IGF1R, with INSR contributing more prominently. These findings, including the largely INSR/IGF1R-independent ERK1/2 activation, provide mechanistic insight into Pas2r12-mediated protein delivery.