Exploring Transferrin-Receptor Interactions at the Single-Molecule Level

Abstract

Interaction between the iron transporter protein transferrin (Tf) and its receptor at the cell surface is fundamental for most living organisms. Tf receptor (TfR) binds iron-loaded Tf (holo-Tf) and transports it to endosomes, where acidic pH favors iron release. Iron-free Tf (apo-Tf) is then brought back to the cell surface and dissociates from TfR. Here we investigated the Tf-TfR interaction at the single-molecule level under different conditions encountered during the Tf cycle. An atomic force microscope tip functionalized with holo-Tf or apo-Tf was used to probe TfR. We tested both purified TfR anchored to a mica substrate and in situ TfR at the surface of living cells. Dynamic force measurements showed similar results for TfR on mica or at the cell surface but revealed striking differences between holo-Tf-TfR and apo-Tf-TfR interactions. First, the forces necessary to unbind holo-Tf and TfR are always stronger compared to the apo-Tf-TfR interaction. Second, dissociation of holo-Tf-TfR complex involves overcoming two energy barriers, whereas the apo-Tf-TfR unbinding pathway comprises only one energy barrier. These results agree with a model that proposes differences in the contact points between holo-Tf-TfR and apo-Tf-TfR interactions.

Noncanonical interactions between serum transferrin and transferrin receptor evaluated with electrospray ionization mass spectrometry

“…The results of earlier cell binding assays suggested that iron removal from hTf results in a dramatic reduction of its receptor affinity (6)(7)(8)(9), while the results of more recent biophysical studies were consistent with a complete loss of the receptorbinding competence for the apo-hTf under conditions mimicking the extra cellular environment (11)(12)(13)(14). ESI MS provides conclusive evidence that aTf has the ability to form relatively stable complexes with TfR at neutral and mildly basic pH.…”

“…However, other studies failed to detect apo-hTf binding to TfR at neutral pH (10), and similar conclusions came from work specifically targeting the hTf/TfR interaction using orthogonal biophysical techniques, such as surface plasmon resonance-based binding assays (11)(12)(13) and atomic force microscopy (14). As a result, the idea of apo-hTf/TfR association at neutral pH became increasingly apocryphal.…”

Profiling TRA-1-81 antigen distribution on a human embryonic stem cell

“…Besides the capability of high resolution topographic imaging, a singular advantage of AFM is the ability of identifying protein species via specific interactions. This principle has been adapted in studies of biomimic surfaces and living cells [10][11][12][13][14]. Here we apply this method in revealing the distribution and association of TRA-1-81 on undifferentiated and differentiated hES cells at the single cell level.…”

Proteínas relacionadas con el metabolismo del hierro corporal

“…La proteína puede almacenar hastaneuroferritinemia con un fenotipo de sobrecarga de hierro en el cerebro, igualmente se puede presentar como hiperferritinemia y las cataratas son la principal complicación (42,43). (45,46). Es considerado el principal captador celular de hierro y se localiza en la membrana del enterocito, del hepatocito, de los eritrocitos, en la placenta, en la médula ósea y en el músculo esquelético (9,11,33,47,48).…”