{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,16]],"date-time":"2025-12-16T05:16:02Z","timestamp":1765862162293,"version":"3.48.0"},"reference-count":34,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,12,12]],"date-time":"2025-12-12T00:00:00Z","timestamp":1765497600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["20K05744"],"award-info":[{"award-number":["20K05744"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["24K08630"],"award-info":[{"award-number":["24K08630"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background: 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. Methods: 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\u2013regulated 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. Results: 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\u2013EGFP 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\u201316%, and IGF1R knockdown to 19\u201365%. In INSR-overexpressing lines, Pas2r12\u2013EGFP induced INSR\/IGF1R phosphorylation (6.0-fold) and enhanced delivery (230\u2013270%). In IGF1R-overexpressing lines, Pas2r12\u2013EGFP did not induce phosphorylation, and delivery decreased to 60\u201369%. Conclusions: 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.<\/jats:p>","DOI":"10.3390\/ph18121885","type":"journal-article","created":{"date-parts":[[2025,12,12]],"date-time":"2025-12-12T15:27:20Z","timestamp":1765553240000},"page":"1885","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide"],"prefix":"10.3390","volume":"18","author":[{"given":"Keito","family":"Sugai","sequence":"first","affiliation":[{"name":"Graduate School of Health Sciences, Niigata University, 2-746 Asahimachi-dori, Chuo-ku, Niigata 951-8518, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3670-8927","authenticated-orcid":false,"given":"Akiko","family":"Okuda","sequence":"additional","affiliation":[{"name":"Graduate School of Health Sciences, Niigata University, 2-746 Asahimachi-dori, Chuo-ku, Niigata 951-8518, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1016\/j.drudis.2014.09.017","article-title":"Therapeutic Applications of the Cell-Penetrating HIV-1 Tat Peptide","volume":"20","author":"Rizzuti","year":"2015","journal-title":"Drug Discov. Today"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"5836","DOI":"10.1074\/jbc.M007540200","article-title":"Arginine-Rich Peptides. An Abundant Source of Membrane-Permeable Peptides Having Potential as Carriers for Intracellular Protein Delivery","volume":"276","author":"Futaki","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"637","DOI":"10.1016\/j.addr.2004.10.007","article-title":"Intracellular Delivery of Large Molecules and Small Particles by Cell-Penetrating Proteins and Peptides","volume":"57","author":"Gupta","year":"2005","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"670","DOI":"10.1111\/j.1582-4934.2007.00062.x","article-title":"Macropinocytosis: Searching for an Endocytic Identity and Role in the Uptake of Cell Penetrating Peptides","volume":"11","year":"2007","journal-title":"J. Cell. Mol. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1038\/nm996","article-title":"Transducible TAT-HA Fusogenic Peptide Enhances Escape of TAT-Fusion Proteins after Lipid Raft Macropinocytosis","volume":"10","author":"Wadia","year":"2004","journal-title":"Nat. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"967","DOI":"10.1016\/j.addr.2003.10.041","article-title":"GALA: A Designed Synthetic PH-Responsive Amphipathic Peptide with Applications in Drug and Gene Delivery","volume":"56","author":"Li","year":"2004","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1290","DOI":"10.1016\/j.bpj.2017.06.053","article-title":"PH-Dependent Membrane Interactions of the Histidine-Rich Cell-Penetrating Peptide LAH4-L1","volume":"113","author":"Wolf","year":"2017","journal-title":"Biophys. J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1849","DOI":"10.1021\/acs.biomac.8b01299","article-title":"Oligoarginine-Bearing Tandem Repeat Penetration-Accelerating Sequence Delivers Protein to Cytosol via Caveolae-Mediated Endocytosis","volume":"20","author":"Okuda","year":"2019","journal-title":"Biomacromolecules"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1016\/j.jconrel.2009.05.019","article-title":"Enhanced Intracellular Delivery Using Arginine-Rich Peptides by the Addition of Penetration Accelerating Sequences (Pas)","volume":"138","author":"Takayama","year":"2009","journal-title":"J. Control. Release"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1222","DOI":"10.1021\/mp200518n","article-title":"Effect of the Attachment of a Penetration Accelerating Sequence and the Influence of Hydrophobicity on Octaarginine-Mediated Intracellular Delivery","volume":"9","author":"Takayama","year":"2012","journal-title":"Mol. Pharm."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1057","DOI":"10.1083\/jcb.150.5.1057","article-title":"Endothelial Cell-Surface Gp60 Activates Vesicle Formation and Trafficking via G(i)-Coupled Src Kinase Signaling Pathway","volume":"150","author":"Minshall","year":"2000","journal-title":"J. Cell Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1111\/j.1600-0854.2009.01008.x","article-title":"Spiral Coating of the Endothelial Caveolar Membranes as Revealed by Electron Tomography and Template Matching","volume":"11","author":"Lebbink","year":"2010","journal-title":"Traffic"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1016\/j.yjmcc.2011.07.007","article-title":"Caveolae Create Local Signalling Domains through Their Distinct Protein Content, Lipid Profile and Morphology","volume":"52","author":"Harvey","year":"2012","journal-title":"J. Mol. Cell. Cardiol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4346","DOI":"10.1093\/emboj\/cdg439","article-title":"Gangliosides Are Receptors for Murine Polyoma Virus and SV40","volume":"22","author":"Tsai","year":"2003","journal-title":"EMBO J."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1016\/j.micron.2006.03.012","article-title":"Regulatory Role of Kinases and Phosphatases on the Internalisation of Caveolae in HepG2 Cells","volume":"38","author":"Botos","year":"2007","journal-title":"Micron"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"152356","DOI":"10.1016\/j.bbrc.2025.152356","article-title":"Phosphoproteomic Analysis of Signal Transduction Dynamics Induced by a Modified Cell-Penetrating Peptide\u2013Protein Complex","volume":"778","author":"Okuda","year":"2025","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Cevenini, A., Orr\u00f9, S., Mancini, A., Alfieri, A., Buono, P., and Imperlini, E. (2018). Molecular Signatures of the Insulin-like Growth Factor 1-Mediated Epithelial-Mesenchymal Transition in Breast, Lung and Gastric Cancers. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19082411"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Thiel, G., Guethlein, L.A., and R\u00f6ssler, O.G. (2021). Insulin-Responsive Transcription Factors. Biomolecules, 11.","DOI":"10.3390\/biom11121886"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Fagerholm, S., \u00d6rtegren, U., Karlsson, M., Ruishalme, I., and Str\u00e5lfors, P. (2009). Rapid Insulin-Dependent Endocytosis of the Insulin Receptor by Caveolae in Primary Adipocytes. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0005985"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Salani, B., Passalacqua, M., Maffioli, S., Briatore, L., Hamoudane, M., Contini, P., Cordera, R., and Maggi, D. (2010). IGF-IR Internalizes with Caveolin-1 and PTRF\/Cavin in HaCat Cells. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0014157"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Lodhia, K., Tienchaiananda, P., and Haluska, P. (2015). Understanding the Key to Targeting the IGF Axis in Cancer: A Biomarker Assessment. Front. Oncol., 5.","DOI":"10.3389\/fonc.2015.00142"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1403206","DOI":"10.1155\/2017\/1403206","article-title":"The Role of Insulin Receptor Isoforms in Diabetes and Its Metabolic and Vascular Complications","volume":"2017","author":"Escribano","year":"2017","journal-title":"J. Diabetes Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"T45","DOI":"10.1530\/JME-17-0250","article-title":"40 YEARS OF IGF1: IGF System in Sarcomas: A Crucial Pathway with Many Unknowns to Exploit for Therapy","volume":"61","author":"Mancarella","year":"2018","journal-title":"J. Mol. Endocrinol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Lee, J.S., Tocheny, C.E., and Shaw, L.M. (2022). The Insulin-like Growth Factor Signaling Pathway in Breast Cancer: An Elusive Therapeutic Target. Life, 12.","DOI":"10.3390\/life12121992"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1573","DOI":"10.1093\/carcin\/bgu070","article-title":"OVA66 Increases Cell Growth, Invasion and Survival via Regulation of IGF-1R\u2013MAPK Signaling in Human Cancer Cells","volume":"35","author":"Rao","year":"2014","journal-title":"Carcinogenesis"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"S33","DOI":"10.1677\/erc.1.01280","article-title":"IGF1R Signalling and Its Inhibition","volume":"13","author":"Riedemann","year":"2006","journal-title":"Endocr. Relat. Cancer"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"a008946","DOI":"10.1101\/cshperspect.a008946","article-title":"The Insulin Receptor: Both a Prototypical and Atypical Receptor Tyrosine Kinase","volume":"5","author":"Hubbard","year":"2013","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Werner, H. (2023). The IGF1 Signaling Pathway: From Basic Concepts to Therapeutic Opportunities. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241914882"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"174588","DOI":"10.1016\/j.ejphar.2021.174588","article-title":"A Review on the Role of Different Ephrins in Glioma","volume":"917","author":"Zhu","year":"2022","journal-title":"Eur. J. Pharmacol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3575","DOI":"10.1111\/bph.12678","article-title":"Lysophospholipid Receptor Nomenclature Review: IUPHAR Review 8","volume":"171","author":"Kihara","year":"2014","journal-title":"Br. J. Pharmacol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1095\/biolreprod.112.100750","article-title":"Compartmentalizing Proximal FGFR1 Signaling in Ovine Placental Artery Endothelial Cell Caveolae","volume":"87","author":"Feng","year":"2012","journal-title":"Biol. Reprod."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1186\/1476-4598-7-69","article-title":"Radiation-Induced Caveolin-1 Associated EGFR Internalization Is Linked with Nuclear EGFR Transport and Activation of DNA-PK","volume":"7","author":"Dittmann","year":"2008","journal-title":"Mol. Cancer"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e70012","DOI":"10.1111\/boc.70012","article-title":"The Different Cellular Entry Routes for Drug Delivery Using Cell Penetrating Peptides","volume":"117","author":"Okafor","year":"2025","journal-title":"Biol. Cell"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"475","DOI":"10.1021\/bc034181g","article-title":"Arginine Carrier Peptide Bearing Ni(II) Chelator to Promote Cellular Uptake of Histidine-Tagged Proteins","volume":"15","author":"Futaki","year":"2004","journal-title":"Bioconjug. Chem."}],"container-title":["Pharmaceuticals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8247\/18\/12\/1885\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,16]],"date-time":"2025-12-16T05:12:49Z","timestamp":1765861969000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8247\/18\/12\/1885"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,12]]},"references-count":34,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["ph18121885"],"URL":"https:\/\/doi.org\/10.3390\/ph18121885","relation":{},"ISSN":["1424-8247"],"issn-type":[{"type":"electronic","value":"1424-8247"}],"subject":[],"published":{"date-parts":[[2025,12,12]]}}}