PMID: 18533687

 

    Legend: Gene, Sites

Title : Identification of the N-linked glycosylation sites of the human relaxin receptor and effect of glycosylation on receptor function

Abstract :
  1. The relaxin receptor , RXFP1 , is a member of the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family
  2. These receptors are characterized by a large extracellular ectodomain containing leucine-rich repeats which contain the primary ligand binding site
  3. RXFP1 contains six putative Asn-linked glycosylation sites in the ectodomain at positions Asn-14, Asn-105, Asn-242, Asn-250, Asn-303, and Asn-346 , which are highly conserved across species
  4. N-Linked glycosylation is the most common post-translational modification of G-protein-coupled receptors , although its role in modulating receptor function differs
  5. We herein investigate the actual N-linked glycosylation status of RXFP1 and the functional ramifications of these post-translational modifications
  6. Site-directed mutagenesis was utilized to generate single- or multiple-glycosylation site mutants of FLAG-tagged human RXFP1 which were then transiently expressed in HEK-293T cells
  7. Glycosylation status was analyzed by immunoprecipitation and Western blot and receptor function analyzed with an anti-FLAG ELISA, (33)P-H2 relaxin competition binding, and cAMP activity measurement
  8. All of the potential N-glycosylation sites of RXFP1 were utilized in HEK-293T cells, and importantly, disruption of glycosylation at individual or combinations of double and triple sites had little effect on relaxin binding
  9. However, combinations of glycosylation sites were required for cell surface expression and cAMP signaling
  10. In particular, N-glycosylation at Asn-303 of RXFP1 was required for optimal intracellular cAMP signaling
  11. Hence, as is the case for other LGR family members, N-glycosylation is essential for the transport of the receptor to the cell surface
  12. Additionally, it is likely that glycosylation is also essential for the conformational changes required for G-protein coupling and subsequent cAMP signaling
Output (sent_index, trigger, protein, sugar, site):
  • 0. glycosylation, , receptor, -, -
  • 0. glycosylation, , receptor, -, sites
  • 1. leucine-rich, , -, the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family, leucine
  • 10. N-glycosylation, , -, -, Asn-303
  • 10. N-glycosylation, , cAMP, -, Asn-303
  • 11. N-glycosylation, , receptor, -, -
  • 2. containing, , -, leucine-rich repeats, ectodomain
  • 2. leucine-rich, , -, leucine-rich repeats, leucine
  • 3. glycosylation, , -, -, positions Asn-14, Asn-105, Asn-242, Asn-250, Asn-303, and Asn-346
  • 3. glycosylation, , -, -, sites
  • 5. glycosylation, , RXFP1, -, -
  • 8. N-glycosylation, , RXFP1, -, sites
  • 8. utilized, , -, -, sites
  • 9. glycosylation, , -, -, sites
Output(Part-Of) (sent_index, protein, site):
  • 0. receptor, sites
  • 10. RXFP1, Asn-303
  • 3. RXFP1, sites
  • 8. RXFP1, sites
*Output_Site_Fusion* (sent_index, protein, sugar, site):
  • 10. cAMP, -, Asn-303
  • 3. RXFP1, -, positions Asn-14, Asn-105, Asn-242, Asn-250, Asn-303, and Asn-346

 

 

Protein NCBI ID SENTENCE INDEX
RXFP1 59350 1,3,5,6,8,10