A domain of the insulin receptor required for endocytosis in rat fibroblasts.
To study the mechanism and role of ligand-dependent endocytosis, we have engineered a mutant insulin receptor that retains its insulin binding and insulin-stimulated tyrosine kinase activities but does not exhibit ligand-induced internalization. The mutant has a deletion of the 16th exon which encodes 22 amino acids (residues 944-965) on the cytoplasmic side of the transmembrane region of the receptor beta-subunit. When the cDNA is transfected in Rat 1 cells, the mutant receptor (HIR delta ex16) is processed to a glycosylated alpha 2 beta 2 heterotetramer and expressed at the cell surface. HIR delta ex16 receptors bind insulin with lower affinity than normal receptors (ED50 for insulin competition = 1.1 nM compared with 0.2 nM for normal receptors), but binding is normal in detergent solution. The mutant HIR delta ex16 receptor undergoes insulin-dependent autophosphorylation and activation as a tyrosine kinase toward exogenous substrates in vitro. In vivo, the receptor is also enzymatically active, as assessed 1) by the ability of antiphosphotyrosine antibodies to precipitate equivalent proportions (58-60%) of occupied wild type or mutant receptors and 2) by immunoblotting extracts of insulin-stimulated cells using antiphosphotyrosine antibodies. In the latter experiment, cells expressing HIR delta ex16 receptors exhibit tyrosine phosphorylation of insulin receptor beta-subunits as well as of pp 185, a putative substrate of the receptor. Despite the ability to bind insulin and activate as a tyrosine kinase, HIR delta ex16 receptors do not internalize in Rat 1 cells. Whereas normal surface receptors covalently labeled with the photoaffinity reagent 125I-NAPA-DP insulin are 36% intracellular after 1 h at 37 degrees C, only background levels of internalization are seen when HIR delta ex16 receptors are labeled. The HIR delta ex16 receptors mediate no internalization or degradation of 125I-insulin compared with control untransfected Rat 1 cells, and they do not down-regulate after long exposure to saturating concentrations of insulin. We conclude that the 16th exon encodes a domain necessary for ligand-dependent endocytosis.
Thies
R S
RS
Department of Medicine, University of California, San Diego, La Jolla 92093.
Webster
N J
NJ
McClain
D A
DA
D001483:Base Sequence; D002460:Cell Line; D002872:Chromosome Deletion; D015536:Down-Regulation; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D007328:Insulin; D015345:Oligonucleotide Probes; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin