C‐mannosylation regulates stabilization of RAMP1 protein and RAMP1‐mediated cell migration

• Published in: The FEBS journal Vol. 290; no. 1; pp. 196 - 208
• Main Authors: Mizuta, Hayato, Takakusaki, Ayane, Suzuki, Takehiro, Otake, Keisuke, Dohmae, Naoshi, Simizu, Siro
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.01.2023
• Subjects: Calcitonin; Cell adhesion & migration; Cell Membrane; Cell migration; Cell Movement; CLR; Cytokine receptors; Cytokines; C‐mannosylation; Dimerization; Glycosylation; Localization; Mannose; Mass spectrometry; Mass spectroscopy; migration; Proteins; RAMP1; Receptor activity modifying proteins; Receptors; Receptors, Cytokine; Stability; Thrombospondin; Thrombospondins; Tryptophan; CLR; migration; C-mannosylation; RAMP1; stability
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.16592

C‐mannosylation is a unique type of protein glycosylation via C‐C linkage between an α‐mannose and a tryptophan residue. This modification has been identified in about 30 proteins and regulates several functions, such as protein secretion and intracellular localization, as well as protein stability. About half of C‐mannosylated proteins are categorized as proteins containing thrombospondin type 1 repeat domain or type I cytokine receptors. To evaluate whether C‐mannosylation broadly affects protein functions regardless of protein domain or family, we have sought to identify other types of C‐mannosylated protein and analyse their functions. In this study, we focused on receptor activity modifying protein 1, which neither contains thrombospondin type 1 repeat domain nor belongs to the type I cytokine receptors. Our mass spectrometry analysis demonstrated that RAMP1 is C‐mannosylated at Trp56. It has been shown that RAMP1 transports to the plasma membrane after dimerization with calcitonin receptor‐like receptor and is important for ligand‐dependent downstream signalling activation. Our results showed that C‐mannosylation has no effect on this transport activity. On the other hand, C‐mannosylation did enhance protein stability and cell migration activity. Our data may provide new insight into both C‐mannosylation research and novel RAMP1 analysis. C‐mannosylation is a unique and rare type of protein glycosylation. In this study, we first demonstrated that RAMP1 is C‐mannosylated at Trp56. We showed that the levels of the heterodimer of RAMP1 with the calcitonin receptor‐like receptor (CLR) at the cell membrane do not differ between mannosylated and non‐mannosylated RAMP1. Consistent with these results, the response to the ligand, CGRP, showed no difference either. However, C‐mannosylation of RAMP1 enhances its stability and cell migration.