Fig. 5

Glycosylation of EREG is crucial for maintaining its membrane subcellular location and autocrine activity. a GFP-EREG localization in HEK293 cells expressing WT, WT + TM, N47Q, or 5NQ mutant EREG by IF staining. b Colocalization of EREG and F-actin in HEK293 cells expressing WT, 5NQ, or N90Q mutant EREG by IF staining. c ELISA of EREG levels in conditioned medium from HEK293 cells expressing WT, N47Q, N90Q, or 2NQ mutant EREG. d HN4 cells were treated for 5 min with CM from HEK293 cells expressing vector, WT, or 5NQ mutant and analyzed by WB with the indicated antibodies. e Protein structure of mutant N47Q. The N and O atoms of the polar uncharged asparagine at position 47 of the wild-type protein form hydrogen bonds with the polar uncharged serine O atom at position 44 and the polar uncharged asparagine O atom at position 90. The hydrogen bond distances are 2.0 and 1.9 Å. The N47Q mutation results in the substitution of the polar uncharged asparagine amino acid at position 47 by the polar uncharged glutamine. After the mutation to glutamine, it forms a hydrogen bond with the non-polar serine O atom at position 44. The hydrogen bond distance is 3.1 Å, and it cannot interact with the N90 amino acid in polarity. The changes in the interaction of these amino acids may lead to changes in the protein structure and functional components after the mutation