A hotspot for enhancing insulin receptor activation revealed by a conformation-specific allosteric aptamer

Abstract

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.

Functional selectivity of insulin receptor revealed by aptamer-trapped receptor structures

“…7c ) 20 , 33 . This indicates that the insulin-bound Γ-shaped structure of IR is more favorable for A43 binding than the apo-IR structure, which explains the mutual positive cooperativity between insulin and A43 23 , 24 , 30 . In contrast to insulin, which exhibits negative cooperativity, A62 and A43 exhibit positive cooperativity for insulin binding to IR 18 , 30 .…”

“…In contrast to A62, the A43 aptamer alone has no agonistic activity, but it acts as a positive allosteric modulator for IR activation in the presence of insulin 30 . Moreover, A43 and insulin binding to IR exhibited mutual positive cooperativity 30 .…”

“…In contrast to A62, the A43 aptamer alone has no agonistic activity, but it acts as a positive allosteric modulator for IR activation in the presence of insulin 30 . Moreover, A43 and insulin binding to IR exhibited mutual positive cooperativity 30 . To understand how A43 stabilizes the insulin-bound IR, we prepared the complex of the IR dimer, A43, and insulin (IR A43+Ins ), and determined the structure at 3.62 Å resolution (Supplementary Fig.…”

“…IR-A62 (A62) is an aptamer agonist for IR that preferentially induces m-pY1150 in the kinase domain and selectively stimulates the AKT pathway and glucose uptake 18 . The IR-A43 aptamer (A43) is a positive allosteric modulator that enhances IR activation by stabilizing insulin binding 30 . Insulin and A43 bind to IR with mutual positive cooperativity, which potentiates IR autophosphorylation and downstream signaling.…”

The Insulin Receptor: An Important Target for the Development of Novel Medicines and Pesticides

“…IR-A48 is an agonistic aptamer that binds to and then activates the IR [ 152 ]. IR-A43 is a sensitizing aptamer that effectively binds to the IR and enhances insulin sensitivity [ 153 , 156 ]. IR-A62 acts as a biased agonist that binds to the extracellular domain of the IR and preferentially induces Y1150 monophosphorylation of IR [ 154 ].…”