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Specific insulin/IGF1 hybrid receptor activation assay reveals IGF1 as a more potent ligand than insulin.

Slaaby R - Sci Rep (2015)

Bottom Line: The hybrid receptor specificity is obtained from a combination of two specific antibodies for IGF1R and for an IR tyrosine phosphorylation site.The specificity was shown by immunoprecipitations and Western blot analysis.It is reported here that both splice variants of insulin/IGF1 receptor hybrids are activated by IGF1 with >20-fold higher potency than insulin.

View Article: PubMed Central - PubMed

Affiliation: Novo Nordisk, Novo Nordisk Park, DK-2760 Måløv, Denmark.

ABSTRACT
This novel method enables specific measurement of the activation of hybrid receptors formed between the Insulin Receptor (IR) and the Insulin-like Growth Factor 1 Receptor (IGF1R). These hybrid receptors are present in tissues and cell lines expressing both IR and IGF1R. It is therefore challenging to separate the homodimer and hybrid receptor activation properties. This ELISA method enabled fast and quantitative measurements of activated hybrid receptors. The hybrid receptor specificity is obtained from a combination of two specific antibodies for IGF1R and for an IR tyrosine phosphorylation site. The specificity was shown by immunoprecipitations and Western blot analysis. IR exists as two splice variants; consequently, two splice variants of hybrid receptors can be expressed. It is reported here that both splice variants of insulin/IGF1 receptor hybrids are activated by IGF1 with >20-fold higher potency than insulin.

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ELISA measurements of specific insulin/IGF1 receptor hybrid activation.Activation of the insulin/IGF1 receptor hybrid Hybrid-A (a) and Hybrid-B (b) measured by detection in ELISA of tyrosine phosphorylation of IRY1334. Concentration–response curves for insulin (Δ) and IGF1 () stimulation (see Methods).
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f2: ELISA measurements of specific insulin/IGF1 receptor hybrid activation.Activation of the insulin/IGF1 receptor hybrid Hybrid-A (a) and Hybrid-B (b) measured by detection in ELISA of tyrosine phosphorylation of IRY1334. Concentration–response curves for insulin (Δ) and IGF1 () stimulation (see Methods).

Mentions: In order to quantitatively compare the activation of insulin/IGF1 receptor hybrids by ligands, an ELISA was developed using the antibody combination described above. The specific hybrid ELISA was created by coating 96 well plates with IGF1R specific 24–31 antibody, with detection using IR pY1334 antibody. The secondary anti-rabbit antibody coupled with horseradish peroxidase ensured a readout of 450 nm after addition of the reagent. Representative concentration–response curves from activation of Hybrid-A and Hybrid-B are shown in Fig. 2a and b. The two splice variants of the hybrid receptors were significantly more responsive to IGF1 than insulin with respective EC50 values for Hybrid-A and Hybrid-B hybrid receptors of 6 ± 3 nM and 12 ± 2 nM with IGF1 and 342 ± 121 nM and 325 ± 88 nM with insulin (Table 1). The maximal insulin response on tyrosine phosphorylation of IR pY1334 reached 93% and 88% of the maximal response of IGF1 for Hybrid-A and Hybrid-B, respectively. In order to test the assay on a widely used cell line, the human Hepatoma cell line (HepG2) was chosen because it is known to contain IR, IGF1R and insulin/IGF1 receptor hybrids23. The activation of hybrid receptors was detected with the ELISA, with EC50 1.9 ± 1.0 nM and 42.4 ± 12.3 nM after IGF1 and insulin stimulation, respectively (Table 1).


Specific insulin/IGF1 hybrid receptor activation assay reveals IGF1 as a more potent ligand than insulin.

Slaaby R - Sci Rep (2015)

ELISA measurements of specific insulin/IGF1 receptor hybrid activation.Activation of the insulin/IGF1 receptor hybrid Hybrid-A (a) and Hybrid-B (b) measured by detection in ELISA of tyrosine phosphorylation of IRY1334. Concentration–response curves for insulin (Δ) and IGF1 () stimulation (see Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300458&req=5

f2: ELISA measurements of specific insulin/IGF1 receptor hybrid activation.Activation of the insulin/IGF1 receptor hybrid Hybrid-A (a) and Hybrid-B (b) measured by detection in ELISA of tyrosine phosphorylation of IRY1334. Concentration–response curves for insulin (Δ) and IGF1 () stimulation (see Methods).
Mentions: In order to quantitatively compare the activation of insulin/IGF1 receptor hybrids by ligands, an ELISA was developed using the antibody combination described above. The specific hybrid ELISA was created by coating 96 well plates with IGF1R specific 24–31 antibody, with detection using IR pY1334 antibody. The secondary anti-rabbit antibody coupled with horseradish peroxidase ensured a readout of 450 nm after addition of the reagent. Representative concentration–response curves from activation of Hybrid-A and Hybrid-B are shown in Fig. 2a and b. The two splice variants of the hybrid receptors were significantly more responsive to IGF1 than insulin with respective EC50 values for Hybrid-A and Hybrid-B hybrid receptors of 6 ± 3 nM and 12 ± 2 nM with IGF1 and 342 ± 121 nM and 325 ± 88 nM with insulin (Table 1). The maximal insulin response on tyrosine phosphorylation of IR pY1334 reached 93% and 88% of the maximal response of IGF1 for Hybrid-A and Hybrid-B, respectively. In order to test the assay on a widely used cell line, the human Hepatoma cell line (HepG2) was chosen because it is known to contain IR, IGF1R and insulin/IGF1 receptor hybrids23. The activation of hybrid receptors was detected with the ELISA, with EC50 1.9 ± 1.0 nM and 42.4 ± 12.3 nM after IGF1 and insulin stimulation, respectively (Table 1).

Bottom Line: The hybrid receptor specificity is obtained from a combination of two specific antibodies for IGF1R and for an IR tyrosine phosphorylation site.The specificity was shown by immunoprecipitations and Western blot analysis.It is reported here that both splice variants of insulin/IGF1 receptor hybrids are activated by IGF1 with >20-fold higher potency than insulin.

View Article: PubMed Central - PubMed

Affiliation: Novo Nordisk, Novo Nordisk Park, DK-2760 Måløv, Denmark.

ABSTRACT
This novel method enables specific measurement of the activation of hybrid receptors formed between the Insulin Receptor (IR) and the Insulin-like Growth Factor 1 Receptor (IGF1R). These hybrid receptors are present in tissues and cell lines expressing both IR and IGF1R. It is therefore challenging to separate the homodimer and hybrid receptor activation properties. This ELISA method enabled fast and quantitative measurements of activated hybrid receptors. The hybrid receptor specificity is obtained from a combination of two specific antibodies for IGF1R and for an IR tyrosine phosphorylation site. The specificity was shown by immunoprecipitations and Western blot analysis. IR exists as two splice variants; consequently, two splice variants of hybrid receptors can be expressed. It is reported here that both splice variants of insulin/IGF1 receptor hybrids are activated by IGF1 with >20-fold higher potency than insulin.

Show MeSH