2 research outputs found
Targeting a GâProtein-Coupled Receptor Overexpressed in Endocrine Tumors by Magnetic Nanoparticles To Induce Cell Death
Nanotherapy using targeted magnetic nanoparticles grafted with peptidic ligands of receptors overexpressed in cancers is a promising therapeutic strategy. However, nanoconjugation of peptides can dramatically affect their properties with respect to receptor recognition, mechanism of internalization, intracellular trafficking, and fate. Furthermore, investigations are needed to better understand the mechanism whereby application of an alternating magnetic field to cells containing targeted nanoparticles induces cell death. Here, we designed a nanoplatform (termed MG-IONP-DY647) composed of an iron oxide nanocrystal decorated with a ligand of a G-protein coupled receptor, the cholecystokinin-2 receptor (CCK2R) that is overexpressed in several malignant cancers. MG-IONP-DY647 did not stimulate inflammasome of Raw 264.7 macrophages. They recognized cells expressing CCK2R with a high specificity, subsequently internalized <i>via</i> a mechanism involving recruitment of β-arrestins, clathrin-coated pits, and dynamin and were directed to lysosomes. Binding and internalization of MG-IONP-DY647 were dependent on the density of the ligand at the nanoparticle surface and were slowed down relative to free ligand. Trafficking of CCK2R internalized with the nanoparticles was slightly modified relative to CCK2R internalized in response to free ligand. Application of an alternating magnetic field to cells containing MG-IONP-DY647 induced apoptosis and cell death through a lysosomal death pathway, demonstrating that cell death is triggered even though nanoparticles of low thermal power are internalized in minute amounts by the cells. Together with pioneer findings using iron oxide nanoparticles targeting tumoral cells expressing epidermal growth factor receptor, these data represent a solid basis for future studies aiming at establishing the proof-of-concept of nanotherapy of cancers using ligand-grafted magnetic nanoparticles specifically internalized <i>via</i> cell surface receptors
Distinct CCKâ2 Receptor Conformations Associated with βâArrestinâ2 Recruitment or PhospholipaseâC Activation Revealed by a Biased Antagonist
Seven-transmembrane receptors (7TMRs), also termed G
protein-coupled
receptors (GPCRs), form the largest class of cell surface membrane
receptors, involving several hundred members in the human genome.
Nearly 30% of marketed pharmacological agents target 7TMRs. 7TMRs
adopt multiple conformations upon agonist binding. Biased agonists,
in contrast to non-biased agonists, are believed to stabilize conformations
preferentially activating either G-protein- or β-arrestin-dependent
signaling pathways. However, proof that cognate conformations of receptors
display structural differences within their binding site where biased
agonism initiates, are still lacking. Here, we show that a non-biased
agonist, cholecystokinin (CCK) induces conformational states of the
CCK2R activating Gq-protein-dependent pathway (CCK2R<sup>G</sup>)
or recruiting β-arrestin2 (CCK2R<sup>β</sup>) that are
pharmacologically and structurally distinct. Two structurally unrelated
antagonists competitively inhibited both pathways. A third ligand
(GV150013X) acted as a high affinity competitive antagonist on CCK2R<sup>G</sup> but was nearly inefficient as inhibitor of CCK2R<sup>β</sup>. Several structural elements on both GV150013X and in CCK2R binding
cavity, which hinder binding of GV150013X only to the CCK2R<sup>β</sup> were identified. At last, proximity between two conserved amino
acids from transmembrane helices 3 and 7 interacting through sulfurâaromatic
interaction was shown to be crucial for selective stabilization of
the CCK2R<sup>β</sup> state. These data establish structural
evidence for distinct conformations of a 7TMR associated with β-arrestin-2
recruitment or G-protein coupling and validate relevance of the design
of biased ligands able to selectively target each functional conformation
of 7TMRs