Comparison of the intracellular trafficking of two alternatively spliced isoforms of pp120, a substrate of the insulin receptor tyrosine kinase.
pp120, a substrate of the insulin receptor tyrosine kinase, is a plasma membrane glycoprotein in the hepatocyte. It is expressed as two spliced isoforms differing by the presence (full length) or absence (truncated) of most of the intracellular domain including all phosphorylation sites. Because the two isoforms differ by their ability to regulate receptor-mediated insulin endocytosis and degradation, we aimed to investigate the cellular basis for this functional difference by comparing their intracellular trafficking. During its intracellular assembly, pp120 is transported from the trans-Golgi network to the sinusoidal domain of the plasma membrane before its final transcytosis to the bile canalicular domain. Because both isoforms are expressed in hepatocytes, we examined their intracellular trafficking in NIH 3T3 fibroblasts individually transfected with each isoform. Pulse-chase experiments demonstrated that most of the newly synthesized full-length isoform reached complete maturation at about 60 min of chase. By contrast, only about 40% of the newly synthesized truncated isoform underwent complete maturation, even at more prolonged chase. Moreover, a significant portion of the truncated isoform appeared to be targeted to lysosomes. Abolishing basal phosphorylation on Ser(503) by cAMP-dependent serine kinase by mutating this residue to alanine was correlated with incomplete maturation of full length pp120 in NIH 3T3 cells and hepatocytes. This finding suggests that the intracellular domain of pp120 contains information that regulates its vectorial sorting from the trans-Golgi network to the plasma membrane.
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Department of Pharmacology and Therapeutics, Medical College of Ohio, Toledo, Ohio 43614, USA.
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D016475:3T3 Cells; D017398:Alternative Splicing; D000818:Animals; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D008099:Liver / Q000166:cytology / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D015815:Cell Adhesion Molecules; D020033:Protein Isoforms; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse