PMC7173346_01
PMC7173346_02
PMC7173346_03
A. Schematic of human IGF1R structural domains and encoding exons. Selected previously reported mutations are indicated. The two new heterozygous mutations are boxed. Abbreviations for domains: leucine-rich domains (L1, L2), cysteine-rich, furin-like domains (CR), fibronectin type III domains (FN1, 2, 3), transmembrane domain (TM), tyrosine kinase catalytic domain (TK) and carboxy-terminus tail (CT). B. IGF1R signal sequence cleavage site. The signal sequence is boxed, with numbering of amino acid residues shown. The 5 critical residues are underlined. Thr28 is the first amino acid of these five critical residues. In wild-type (WT), the cleavage site is known and accurately predicted in silico by http://www.cbs.dtu.dk/services/SignalP/. The predicted probability of cleavage is indicated for both WT and the p.Thr28del.
nihms-1061728-f0001.jpg
PMC7173346_03_nihms-1061728-f0001.jpg
F1
IGF1R were transfected into HEK293 cells. Immunoblot analyses indicated that both the p.V1013F and p.Thr28del variants caused significant decreases in IGF1R expression with concomitant poor responses to IGF-I stimulation (Figure 2), thus supporting the pathogenic impact of the variants. It is of note that pIGF1R was detected with increasing loading of p.Thr28del cell lysates, but not in response to IGF-I (Supplementary Figure 1).
A. Western blot analysis of protein cell lysate (30ug) from transfected HEK293 cells untreated and treated with IGF-1. Antibodies for phosphorylated IGFIRP (pIGFIRbeta), total IGFIRP (tIGFIRbeta) and alpha-Tubulin were employed. Wild-type control has a robust phosphorylation response (pIGFIRbeta) to IGF-1 treatment. V1013F and Thr28del show little to no pIGFIRbeta expression and the response to IGF-1 is abrogated. tIGFIRbeta expression is significantly lower in both mutant variants compared to wild-type control. B. Densitometric analysis of average tIGFIR expression relative to alpha-tubulin. *p < 0.05, **p < 0.001.
nihms-1061728-f0002.jpg
PMC7173346_03_nihms-1061728-f0002.jpg
F2
IGF1R were transfected into HEK293 cells. Immunoblot analyses indicated that both the p.V1013F and p.Thr28del variants caused significant decreases in IGF1R expression with concomitant poor responses to IGF-I stimulation (Figure 2), thus supporting the pathogenic impact of the variants. It is of note that pIGF1R was detected with increasing loading of p.Thr28del cell lysates, but not in response to IGF-I (Supplementary Figure 1).