Aptamers are single-stranded oligonucleotides that bind to a specific target with high affinity, and are widely applied in biomedical diagnostics and drug development. However, the use of aptamers has largely been limited to simple binders or inhibitors that interfere with the function of a target protein. Here, we show that an aptamer can also act as a positive allosteric modulator that enhances the activation of a receptor by stabilizing the binding of a ligand to that receptor. We developed an aptamer, named IR-A43, which binds to the insulin receptor, and confirmed that IR-A43 and insulin bind to the insulin receptor with mutual positive cooperativity. IR-A43 alone is inactive, but, in the presence of insulin, it potentiates autophosphorylation and downstream signaling of the insulin receptor. By using the species-specific activity of IR-A43 at the human insulin receptor, we demonstrate that residue Q272 in the cysteine-rich domain is directly involved in the insulin-enhancing activity of IR-A43. Therefore, we propose that the region containing residue Q272 is a hotspot that can be used to enhance insulin receptor activation. Moreover, our study implies that aptamers are promising reagents for the development of allosteric modulators that discriminate a specific conformation of a target receptor.
Aptamers are widely used as binders that interact with targets with high affinity or as inhibitors of the function of target molecules. However, they have also been used to modulate target protein function, which they achieve by activating the target or stabilizing its conformation. Here, we report a unique aptamer modulator of the insulin receptor (IR), IR-A62. Alone, IR-A62 acts as a biased agonist that preferentially induces Y1150 monophosphorylation of IR. However, when administered alongside insulin, IR-A62 shows variable binding cooperativity depending on the ligand concentration. At low concentrations, IR-A62 acts as a positive allosteric modulator (PAM) agonist that enhances insulin binding, but at high concentrations, it acts as a negative allosteric modulator (NAM) agonist that competes with insulin for IR. Moreover, the concentration of insulin affects the binding of IR-A62 to IR. Finally, the subcutaneous administration of IR-A62 to diabetic mice reduces blood glucose levels with a longer-lasting effect than insulin administration. These findings imply that aptamers can elicit various responses from receptors beyond those of a simple agonist or inhibitor. We expect further studies of IR-A62 to help reveal the mechanism of IR activation and greatly expand the range of therapeutic applications of aptamers.
Insulin is a key regulator of energy metabolism in peripheral tissues but also functions as a growth factor. Insulin binding to the insulin receptor (IR) leads to autophosphorylation of intracellular tyrosine residues, which simultaneously initiates a multitude of signals and functions. In contrast, some artificial (non‐insulin) ligands for the IR result in biased agonism, selectively activating the PI3K/AKT pathway and metabolic effects without activating mitogen‐activated protein kinase (MAPK) pathway and mitogenic effects. However, the precise mechanism of biased agonism at the receptor level remains unclear. The biased agonist IR‐A48 aptamer selectively induces mono‐phosphorylation of Tyr1150 residue (m‐pY1150) in the kinase domain of IR. Hence, we hypothesized that IR autophosphorylation is a stepwise process in which formation of m‐pY1150 represents an intermediate step. To explore this idea, we used hybrid receptors in which insulin can bind only one of the two binding sites of the dimeric receptor. Asymmetric insulin binding selectively induced symmetric m‐pY1150 in both kinase domains. Moreover, the juxtamembrane domain, which interacts with the kinase domain, restricted the full activation of IR, and symmetric m‐pY1150 played a crucial role in the rearrangement of intracellular domains to release this restriction. Our findings demonstrate that the symmetry of insulin binding to a dimeric receptor determines the stepwise autophosphorylation of IR. Furthermore, considering that the degree of ligand symmetry on a receptor depends mainly on ligand concentration, our results suggest that IR may play metabolic‐biased roles in peripheral tissues dependent on local insulin concentrations. Support or Funding Information This research was supported by the Global Research Laboratory (GRL) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (No. NRF‐2016K1A1A2912722).
Aptamers are single‐stranded oligonucleotides (DNA or RNA) and form a unique three‐dimensional (3D) structure based on its sequence. These aptamers have high affinity and specificity to target molecule and recognize 3D structure of target molecule surface. For this reason, aptamers have been in the spotlight as a universal capture. Especially, aptamers can modulate target protein as allosteric agonist or allosteric antagonist. However, aptamer‐based allosteric enhancer or sensitizer (hereafter ‐ sensitizer) has not been elucidated. Here, for the first time, we show the INSR‐A43, which is aptamer‐based sensitizer for the insulin receptor. Because insulin receptor plays a key role in regulation of glucose metabolism, it is important to specifically modulate the insulin receptor. INSR‐A43 binds to allosteric site of the insulin receptor and then increases insulin binding to the insulin receptor. Taken together, INSR‐A43 effectively enhances insulin sensitivity to the insulin receptor and sensitizes insulin receptor and insulin receptor‐related downstream signaling pathways. We expect that aptamers can be applied as an attractive tool for receptor sensitization. Support or Funding Information This research was supported by Grobal Research Laboratory (GRL) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (No. NRF‐2016K1A1A2912722)
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