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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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Specific insulin/IGF1 receptor hybrid activation assay.Western blot analysis of immunoprecipitations (a, b and c) with 24–31 (lanes 1–12) and with 83–7 (lanes 13 and 14) on lysates from BHK cells expressing IR-A and IGF1R (lanes 1–8), IR-A (lanes 9, 10, 13 and 14) or IGF1R (lanes 11 and 12). Cell lysate analysis (d, e and f). The blots were probed with IR pY1334 (a and d), IGF1R N-20 (b and e) and IR C-19 (c and f). Cells were either unstimulated (lanes 1 and 5) or stimulated with 1, 10 or 100 nM insulin (lanes 2, 3 and 4), with IGF1 (lanes 6, 7, and 8), with 100 nM insulin (lanes 9, 11 and 13) or with 100 nM IGF1 (lanes 10, 12 and 14) for 30 minutes at 37°C. The data shown is one representative of four independent experiments. BHK, baby hamster kidney; IGF1, Insulin-like Growth Factor 1; IGF1R, Insulin-like Growth Factor 1 Receptor; IR, Insulin Receptor; IP, ImmunoPrecipitation.
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f1: Specific insulin/IGF1 receptor hybrid activation assay.Western blot analysis of immunoprecipitations (a, b and c) with 24–31 (lanes 1–12) and with 83–7 (lanes 13 and 14) on lysates from BHK cells expressing IR-A and IGF1R (lanes 1–8), IR-A (lanes 9, 10, 13 and 14) or IGF1R (lanes 11 and 12). Cell lysate analysis (d, e and f). The blots were probed with IR pY1334 (a and d), IGF1R N-20 (b and e) and IR C-19 (c and f). Cells were either unstimulated (lanes 1 and 5) or stimulated with 1, 10 or 100 nM insulin (lanes 2, 3 and 4), with IGF1 (lanes 6, 7, and 8), with 100 nM insulin (lanes 9, 11 and 13) or with 100 nM IGF1 (lanes 10, 12 and 14) for 30 minutes at 37°C. The data shown is one representative of four independent experiments. BHK, baby hamster kidney; IGF1, Insulin-like Growth Factor 1; IGF1R, Insulin-like Growth Factor 1 Receptor; IR, Insulin Receptor; IP, ImmunoPrecipitation.

Mentions: In order to study whether the antibody combination of IGF1R 24–31 and IR pY1334 could be used in a specific hybrid receptor-activation assay, immunoprecipitation and SDS-PAGE analysis were first performed (Fig. 1). Lysates were prepared from baby hamster kidney (BHK) cells co-expressing IGF1R and IR-A, which were either unstimulated or stimulated with 1, 10 or 100 nM insulin (lanes 1–4) or IGF1 (lanes 5–8) for 30 minutes. Control cells overexpressing only IR-A (lanes 9, 10, 13 and 14) or IGF1R (lanes 11 and 12) were stimulated with 100 nM insulin or IGF1. Two blots for analysis were made; one for immunoprecipitated receptors (Fig. 1a, b and c) and one for the cell lysates that were the input for the immunoprecipitations (Fig. 1d, e and f). In this study, the blots were reprobed, taking advantage of differences in migration within the gel (i.e. the alpha subunit at 120 kDa and the beta subunit at 97 kDa). Hybrid activation was detected in the immunoprecipitated cell lysate with the specific IGF1R antibody 24–31 and Western blot analysis using the specific antibody IR pY1334 (Fig. 1a, lanes 1–8). There was a tendency for the sensitivity to IGF1 to be greater (Fig. 1a, lanes 5–8) than to insulin (Fig. 1a, lanes 1–4). This method is a specific hybrid receptor-activation assay, since co-expression of IR and IGF1R was needed to detect the IR pY1334 of immunoprecipitated IGF1R. The method distinguishes between homo and hetero dimer receptors activation. No signal was detected from the cell line, overexpressing only IGF1R (Fig. 1a, lanes 11 and 12) and from cells expressing only IR-A, no IGF1R was immunoprecipitated (Fig. 1b, lanes 9 and 10), hence IR could not be immunoprecipitated as part of a hybrid receptor. This also proved the specificity of the IGF1R 24–31 antibody. The antibody IR pY1334 can detect activated IR since it gave a strong signal when IR was immunoprecipitated with the IR-specific antibody 83–7 after stimulation with insulin or IGF1 (Fig. 1a, lanes 13 and 14). In the lysates from BHK cells overexpressing both IR and IGF1R (Fig. 1d, lanes 1–4), tyrosine phosphorylation of IR pY1334 was detected even at low doses of insulin. This signal was primarily from homodimer IR rather than hybrid receptors because this high sensitivity to insulin was not detected after immunoprecipitating with IGF1R antibody 24–31 (Fig. 1a, lanes 1–4). The difference in the dose response to IGF1 and insulin was not caused by different expression levels or variations in immunoprecipitations since equal levels of IGF1R were detected in all relevant lanes (Fig. 1b and e) at 120 kDa with the IGF1R alpha subunit antibody N-20. The Western blot was reprobed and the bleed through from the pY1334 probing from panel A or D was detected at 97 kDa. Similarly, an equal IR level was present in all relevant lanes detected, with C-19 antibody recognising the 97 kDa beta subunit (Fig. 1c and f). The bleed through from the N-20 blot was detected at 120 kDa.


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

Slaaby R - Sci Rep (2015)

Specific insulin/IGF1 receptor hybrid activation assay.Western blot analysis of immunoprecipitations (a, b and c) with 24–31 (lanes 1–12) and with 83–7 (lanes 13 and 14) on lysates from BHK cells expressing IR-A and IGF1R (lanes 1–8), IR-A (lanes 9, 10, 13 and 14) or IGF1R (lanes 11 and 12). Cell lysate analysis (d, e and f). The blots were probed with IR pY1334 (a and d), IGF1R N-20 (b and e) and IR C-19 (c and f). Cells were either unstimulated (lanes 1 and 5) or stimulated with 1, 10 or 100 nM insulin (lanes 2, 3 and 4), with IGF1 (lanes 6, 7, and 8), with 100 nM insulin (lanes 9, 11 and 13) or with 100 nM IGF1 (lanes 10, 12 and 14) for 30 minutes at 37°C. The data shown is one representative of four independent experiments. BHK, baby hamster kidney; IGF1, Insulin-like Growth Factor 1; IGF1R, Insulin-like Growth Factor 1 Receptor; IR, Insulin Receptor; IP, ImmunoPrecipitation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: Specific insulin/IGF1 receptor hybrid activation assay.Western blot analysis of immunoprecipitations (a, b and c) with 24–31 (lanes 1–12) and with 83–7 (lanes 13 and 14) on lysates from BHK cells expressing IR-A and IGF1R (lanes 1–8), IR-A (lanes 9, 10, 13 and 14) or IGF1R (lanes 11 and 12). Cell lysate analysis (d, e and f). The blots were probed with IR pY1334 (a and d), IGF1R N-20 (b and e) and IR C-19 (c and f). Cells were either unstimulated (lanes 1 and 5) or stimulated with 1, 10 or 100 nM insulin (lanes 2, 3 and 4), with IGF1 (lanes 6, 7, and 8), with 100 nM insulin (lanes 9, 11 and 13) or with 100 nM IGF1 (lanes 10, 12 and 14) for 30 minutes at 37°C. The data shown is one representative of four independent experiments. BHK, baby hamster kidney; IGF1, Insulin-like Growth Factor 1; IGF1R, Insulin-like Growth Factor 1 Receptor; IR, Insulin Receptor; IP, ImmunoPrecipitation.
Mentions: In order to study whether the antibody combination of IGF1R 24–31 and IR pY1334 could be used in a specific hybrid receptor-activation assay, immunoprecipitation and SDS-PAGE analysis were first performed (Fig. 1). Lysates were prepared from baby hamster kidney (BHK) cells co-expressing IGF1R and IR-A, which were either unstimulated or stimulated with 1, 10 or 100 nM insulin (lanes 1–4) or IGF1 (lanes 5–8) for 30 minutes. Control cells overexpressing only IR-A (lanes 9, 10, 13 and 14) or IGF1R (lanes 11 and 12) were stimulated with 100 nM insulin or IGF1. Two blots for analysis were made; one for immunoprecipitated receptors (Fig. 1a, b and c) and one for the cell lysates that were the input for the immunoprecipitations (Fig. 1d, e and f). In this study, the blots were reprobed, taking advantage of differences in migration within the gel (i.e. the alpha subunit at 120 kDa and the beta subunit at 97 kDa). Hybrid activation was detected in the immunoprecipitated cell lysate with the specific IGF1R antibody 24–31 and Western blot analysis using the specific antibody IR pY1334 (Fig. 1a, lanes 1–8). There was a tendency for the sensitivity to IGF1 to be greater (Fig. 1a, lanes 5–8) than to insulin (Fig. 1a, lanes 1–4). This method is a specific hybrid receptor-activation assay, since co-expression of IR and IGF1R was needed to detect the IR pY1334 of immunoprecipitated IGF1R. The method distinguishes between homo and hetero dimer receptors activation. No signal was detected from the cell line, overexpressing only IGF1R (Fig. 1a, lanes 11 and 12) and from cells expressing only IR-A, no IGF1R was immunoprecipitated (Fig. 1b, lanes 9 and 10), hence IR could not be immunoprecipitated as part of a hybrid receptor. This also proved the specificity of the IGF1R 24–31 antibody. The antibody IR pY1334 can detect activated IR since it gave a strong signal when IR was immunoprecipitated with the IR-specific antibody 83–7 after stimulation with insulin or IGF1 (Fig. 1a, lanes 13 and 14). In the lysates from BHK cells overexpressing both IR and IGF1R (Fig. 1d, lanes 1–4), tyrosine phosphorylation of IR pY1334 was detected even at low doses of insulin. This signal was primarily from homodimer IR rather than hybrid receptors because this high sensitivity to insulin was not detected after immunoprecipitating with IGF1R antibody 24–31 (Fig. 1a, lanes 1–4). The difference in the dose response to IGF1 and insulin was not caused by different expression levels or variations in immunoprecipitations since equal levels of IGF1R were detected in all relevant lanes (Fig. 1b and e) at 120 kDa with the IGF1R alpha subunit antibody N-20. The Western blot was reprobed and the bleed through from the pY1334 probing from panel A or D was detected at 97 kDa. Similarly, an equal IR level was present in all relevant lanes detected, with C-19 antibody recognising the 97 kDa beta subunit (Fig. 1c and f). The bleed through from the N-20 blot was detected at 120 kDa.

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