Characterization of insulin/IGF hybrid receptors: contributions of the insulin receptor L2 and Fn1 domains and the alternatively spliced exon 11 sequence to ligand binding and receptor activation

Abstract

The IR (insulin receptor) and IGFR (type I insulin-like growth factor receptor) are found as homodimers, but the respective pro-receptors can also heterodimerize to form insulin-IGF hybrid receptors. There are conflicting data on the ligand affinity of hybrids, and especially on the influence of different IR isoforms. To investigate further the contribution of individual ligand binding epitopes to affinity and specificity in the IR/IGFR family, we generated hybrids incorporating both IR isoforms (A and B) and IR/IGFR domain-swap chimaeras, by ectopic co-expression of receptor constructs in Chinese hamster ovary cells, and studied ligand binding using both radioligand competition and bioluminescence resonance energy transfer assays. We found that IR-A-IGFR and IR-B-IGFR hybrids bound insulin with similar relatively low affinity, which was intermediate between that of homodimeric IR and homodimeric IGFR. However, both IR-A-IGFR and IR-B-IGFR hybrids bound IGF-I and IGF-II with high affinity, at a level comparable with homodimeric IGFR. Incorporation of a significant fraction of either IR-A or IR-B into hybrids resulted in abrogation of insulin- but not IGF-I-stimulated autophosphorylation. We conclude that the sequence of 12 amino acids encoded by exon 11 of the IR gene has little or no effect on ligand binding and activation of IR-IGFR hybrids, and that hybrid receptors bind IGFs but not insulin at physiological concentrations regardless of the IR isoform they contained. To reconstitute high affinity insulin binding within a hybrid receptor, chimaeras in which the IGFR L1 or L2 domains had been replaced by equivalent IR domains were co-expressed with full-length IR-A or IR-B. In the context of an IR-A-IGFR hybrid, replacement of IR residues 325-524 (containing the L2 domain and part of the first fibronectin domain) with the corresponding IGFR sequence increased the affinity for insulin by 20-fold. We conclude that the L2 and/or first fibronectin domains of IR contribute in trans with the L1 domain to create a high affinity insulin-binding site within a dimeric receptor.

Figure 1. Ligand binding to IR-A and IR-B homodimers

IR-A and IR-B expressed in CHO cells were immunocaptured using anti-IR antibody 83-7 and binding of ¹²⁵I-insulin (50 pM) was measured in the presence of increasing concentrations of unlabelled insulin (A), unlabelled IGF-I (B), or unlabelled IGF-II (C). Binding to IR-A (○) and IR-B (●) is expressed as a percentage of the value in the absence of unlabelled ligand. Data points are the means±S.E.M. for three independent experiments in (A) and triplicate samples within a representative experiment in (B) and (C).

Figure 2. Ligand binding to IGFR homodimers

IGF-I receptors expressed in NIH3T3 cells were immunocaptured using anti-IGFR antibody 17-69 and binding of ¹²⁵I-IGF-I (25 pM) was measured in the presence of increasing concentrations of unlabelled insulin (○) or IGF-I (●). Binding is expressed as a percentage of the value in the absence of unlabelled ligand. Data points are the means±S.E.M. for triplicate samples within a representative experiment.

Figure 3. Ligand binding to IR-A–IGFR and IR-B–IGFR hybrids

Hybrid receptors IR-A–IGFR and IR-B–IGFR were immunocaptured using IR-specific antibody 83-7 and the binding of ¹²⁵I-IGF-I (25 pM) was determined in the presence of unlabelled insulin (A), unlabelled IGF-I (B) or unlabelled IGF-II (C). Binding to IR-A–IGFR (○) and IR-B–IGFR (●) is expressed as a percentage of the value in the absence of unlabelled ligand. Data points are the means±S.E.M. for triplicate samples within a representative experiment in (A) and (C) and three independent experiments in (B).

Figure 4. Effect of insulin on BRET signal of IR-B homodimers and IR-A/IR-B heterodimers

Lysates of cells co-expressing IR-B–luc with either IR-B–YFP (A) or IR-A–GFP (B) were incubated with insulin and the BRET ratio was measured as described in the Experimental section. The results are shown as means±ranges for two independent experiments.

Figure 5. Effects of insulin and IGF-I on BRET signal of IGFR and IR–IGFR hybrids

Lysates of cells co-expressing IGFR–luc with IGFR–YFP, IR-A–GFP or IR-B–YFP were incubated with insulin (A) or IGF-I (B) and the BRET ratio was measured as described in the Experimental section. Results are means±ranges for two independent experiments each conducted in duplicate.

Figure 6. Phosphorylation of IR-A in homodimers and in hybrids

CHO cells were transfected with 40–ng of IR-A cDNA or 1–μg of IGFR plus 0.2–μg of IR-A cDNAs. The cells were stimulated with 10–nM insulin or IGF-I and lysates were immunoprecipitated (IP) with either anti-IR 83-14 (αIR) or anti-IGFR 17-69 (αIGFR) and resolved by SDS/PAGE. (A) Immunoblots (IB) were probed with anti-phosphotyrosine (pY) antibody (4G10). The membrane was then stripped and reprobed with anti-IR antibody (rabbit polyclonal recognizing the C-terminal domain). The blots shown are representative of three independent experiments. (B) Anti-phosphotyrosine blots [as in (A)] were scanned and quantified using MacBAS V2.2, and values were normalized for receptor expression as determined by scanning the anti-IR blots. The results are means±S.E.M. for three independent experiments. Insulin-stimulated phosphorylation was significantly less (*P<0.01) in presence of IGFR (lanes 5 and 6) compared with IR alone (lane 4) as assessed by two-tailed paired Student's t test.

Figure 7. Phosphorylation of IR-B in homodimers and in hybrids

CHO cells were transfected with 80–ng of IR-B cDNA or 1–μg of IGFR plus 0.4–μg of IR-B cDNAs. The cells were stimulated with 10–nM insulin or IGF-I and lysates were immunoprecipitated with either anti-IR 83-14 (αIR) or anti-IGFR 17-69 (αIGFR) and resolved by SDS/PAGE. (A) Immunoblots were probed with anti-phosphotyrosine antibody (4G10). The membrane was then stripped and reprobed with an anti-IR antibody. The blots shown are representative of three independent experiments. (B) Anti-phosphotyrosine blots [as in (A)] were scanned and quantified using MacBAS V2.2, and values were normalized for receptor expression as determined by scanning the anti-IR blots. The results are means±S.E.M. for three independent experiments. Insulin-stimulated phosphorylation was significantly less (*P<0.02) in the presence of IGFR (lanes 5 and 6) compared with IR alone (lane 4) as assessed by two-tailed paired Student's t test.

Figure 8. Reconstitution of high affinity insulin-binding using chimaeric receptors

(A) Schematic representation of the IR, IGFR and IGFR domain swap chimaeras (as previously described in [35]). Domains are indicated as L1, CR (cysteine-rich), L2, Fn1, Fn2, Fn3 (extracellular) and TK (intracellular), with IR (open bars) and IGFR (grey bars). (B) IR and chimaeric IGFR constructs were co-expressed in CHO cells as described in the Experimental section. Cells were lysed, hybrid receptors were immunocaptured using anti-IR antibody 83-7 and binding of ¹²⁵I-IGF1 (25 pM) was measured in the presence of unlabelled insulin. Binding is expressed as percentage of the value in the absence of unlabelled ligand. Data points are the means±S.E.M. of triplicate samples within a representative experiment.

Scheme 1. Model of ligand binding to hybrid receptors

The model is based on that of DeMeyts and Whittaker [11] and assumes that only a single molecule of ligand binds with high affinity to homodimeric IR or IGFR, by contacting sites on both α-subunits, of which site 1 contributes the greater fraction of binding energy. (A) and (B) represent binding of insulin and IGF to homodimeric IR and IGFR respectively. (C) and (D) represent two potential modes of binding to hybrid receptors, in which site 1 is contributed by the IR or IGFR half respectively. (E) and (F) represent the presumed binding modes of labelled insulin and IGF-I respectively, illustrating that IC₅₀ values for competition by unlabelled insulin will be different for the two labelled ligands. The EC₅₀ for receptor activation by insulin correlates with the IC₅₀ for labelled IGF, suggesting that of the two possible insulin binding modes (D) may be more effective than (C) in leading to receptor activation.