Comparison of the intracellular trafficking of two alternatively spliced isoforms of pp120, a substrate of the insulin receptor tyrosine kinase

Abstract

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.

The cytoplasmic domain of CEACAM1-L controls its lateral localization and the organization of desmosomes in polarized epithelial cells

“…However, were the Lys residues mutated to three Ala residues a complete re-localization of CEACAM1-L to the apical surface occurred. These three Lys residues therefore play an important role in the steady state lateral localization of CEACAM1-L. Another important amino acid for CEACAM1-L intracellular transport and function, is Ser503 (Choice et al, 1999;Fournès et al, 2001) and thus we investigated its role in lateral localization of CEACAM1-L by changing it to Ala. This mutation resulted in the complete disappearance of CEACAM1-L from the lateral surface.…”

Sucrase-?-dextrinase in the spontaneously diabetic BioBreed Wistar rat: Altered intracellular carbohydrate processing

“…Absence of an absolute requirement for N-linked glycosylation for transfer of S-D to the surface membrane has been shown in intestinal extracts [Danielsen and Cowell, 1986]. Similarly, complete glycosylation of the insulin receptor, of its close relative, the insulin-like growth factor receptor, and of one of its substrate, pp120, was not required for their insertion in the plasma membrane [Duronio et al, 1986;Choice et al, 1999].…”

“…The S-D immunoprecipitates were boiled for 5 min in 150 mM sodium citrate, 2% SDS, pH 5.5, centrifuged at 15,000g for 5 min and treated with Endo-b-N-acetylglucosaminidase H (Endo H, Boehringer Mannheim Co.), as described previously [Choice et al, 1999]. Brie¯y, Endoglycosidase F/N-glycosidase F Endo H (10 mU) was added to the recovered supernatant, and the digestion mixture placed at 37 C for 1 h with a thin overlay of toluene.…”

“…Treatment of the S-D pellet by Endoglycosidase F/N-glycosidase F (Endo F) (PNGase F, Boerhinger Mannheim Co.) was performed as described previously [Choice et al, 1999]. Brie¯y, the S-D immune pellet ($20 mU of S from ER-Golgi or $200 mU from brush border) was washed three times with 62.5 mM Tris± HCl (pH 6.8), and heated at 100 C for 2 min in 100 ml of 2% SDS, 2% b-mercaptoethanol, 0.2 M sodium phosphate (pH 8.6).…”

The Role of Placental Homeobox Genes in Human Fetal Growth Restriction

“…These genes are potential candidates for regulating epithelial-mesenchymal cell interactions in the human placenta. These genes are also expressed in the embryo and play major roles in embryonic development [78, 79]. Microarray expression profiling of placental trophoblast and endothelial cells revealed that novel placental homeobox genes TGIF, MEIS2E, LIM2 , and SMAP31-12 are also highly expressed in trophoblast cells (Murthi et al unpublished data).…”