Endocytosis and intracellular dissociation rates of human insulin-insulin receptor complexes by quantum dots in living cells.
Insulin signaling is involved in glucose metabolism, cellular growth, and differentiation. Its function is altered in diabetes and many cancer types. Insulin binding to insulin receptor (IR) triggers diverse signaling pathways. However, signal transduction by IR is not mediated exclusively at the cell surface. Activated ligand-receptor complexes are internalized into endosomes from which the IR recruits adapters acting on substrates that are distinct from those accessible at the membrane. We report the biotinylation of human-recombinant insulin (rhIns) specifically at the position 29 of the B chain. We combined visible fluorescent proteins fused to IR and biotinylated rhIns conjugated with streptavidin-quantum dots to perform extended, quantitative experiments in real time. Modified rhIns bound to the IR and conjugated with the quantum dots was internalized with a rate constant (k) of 0.009 min(-1). Dissociation of insulin-IR complex in endocytosed vesicles occurred with k = 0.006 min(-1).
Departamento de Química Biológica, IQUIBICEN-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Intendente Güiraldes 2160, Ciudad Universitaria, C1428EGA, Buenos Aires, Argentina.
Jimena
J
Giudice
Elizabeth A
EA
Jares-Erijman
Federico Coluccio
FC
Leskow
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D004705:Endocytosis / Q000502:physiology*; D057809:HEK293 Cells; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D045663:Quantum Dots; D011972:Receptor, Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D015703:Antigens, CD; C435941:INSR protein, human; D011972:Receptor, Insulin