Structure-function analysis of RAMP1 by alanine mutagenesis

John Simms, Debbie L Hay, Richard J Bailey, Galina Konycheva, Graham Bailey, Mark Wheatley, David R Poyner

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Receptor activity modifying protein 1 (RAMP1) is an integral component of several receptors including the calcitonin gene-related peptide (CGRP) receptor. It forms a complex with the calcitonin receptor-like receptor (CLR) and is required for receptor trafficking and ligand binding. The N-terminus of RAMP1 comprises three helices. The current study investigated regions of RAMP1 important for CGRP or CLR interactions by alanine mutagenesis. Modeling suggested the second and third helices were important in protein-protein interactions. Most of the conserved residues in the N-terminus (M48, W56, Y66, P85, N66, H97, F101, D113, P114, P115), together with a further 13 residues spread throughout three helices of RAMP1, were mutated to alanine and coexpressed with CLR in Cos 7 cells. None of the mutations significantly reduced RAMP expression. Of the nine mutants from helix 1, only M48A had any effect, producing a modest reduction in trafficking of CLR to the cell surface. In helix 2 Y66A almost completely abolished CLR trafficking; L69A and T73A reduced the potency of CGRP to produce cAMP. In helix 3, H97A abolished CLR trafficking; P85A, N86A, and F101A had caused modest reductions in CLR trafficking and also reduced the potency of CGRP on cAMP production. F93A caused a modest reduction in CLR trafficking alone and L94A increased cAMP production. The data are consistent with a CLR recognition site particularly involving Y66 and H97, with lesser roles for adjacent residues in helix 3. L69 and T73 may contribute to a CGRP recognition site in helix 2 also involving nearby residues.

Original languageEnglish
Pages (from-to)198-205
Number of pages8
JournalBiochemistry
Volume48
Issue number1
DOIs
Publication statusPublished - 13 Jan 2009
Externally publishedYes

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Receptor Activity-Modifying Protein 1
Calcitonin Receptor-Like Protein
Mutagenesis
Alanine
Calcitonin Gene-Related Peptide
Calcitonin Gene-Related Peptide Receptors
Proteins

Keywords

  • Adrenomedullin
  • Alanine
  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Calcitonin Gene-Related Peptide
  • Calcitonin Receptor-Like Protein
  • Cercopithecus aethiops
  • Conserved Sequence
  • Cyclic AMP
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Protein Structure, Secondary
  • Protein Transport
  • Receptor Activity-Modifying Protein 1
  • Receptor Activity-Modifying Proteins
  • Receptors, Calcitonin
  • Receptors, Calcitonin Gene-Related Peptide
  • Structure-Activity Relationship
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

Simms, J., Hay, D. L., Bailey, R. J., Konycheva, G., Bailey, G., Wheatley, M., & Poyner, D. R. (2009). Structure-function analysis of RAMP1 by alanine mutagenesis. Biochemistry, 48(1), 198-205. https://doi.org/10.1021/bi801869n

Structure-function analysis of RAMP1 by alanine mutagenesis. / Simms, John; Hay, Debbie L; Bailey, Richard J; Konycheva, Galina; Bailey, Graham; Wheatley, Mark; Poyner, David R.

In: Biochemistry, Vol. 48, No. 1, 13.01.2009, p. 198-205.

Research output: Contribution to journalArticle

Simms, J, Hay, DL, Bailey, RJ, Konycheva, G, Bailey, G, Wheatley, M & Poyner, DR 2009, 'Structure-function analysis of RAMP1 by alanine mutagenesis' Biochemistry, vol. 48, no. 1, pp. 198-205. https://doi.org/10.1021/bi801869n
Simms J, Hay DL, Bailey RJ, Konycheva G, Bailey G, Wheatley M et al. Structure-function analysis of RAMP1 by alanine mutagenesis. Biochemistry. 2009 Jan 13;48(1):198-205. https://doi.org/10.1021/bi801869n
Simms, John ; Hay, Debbie L ; Bailey, Richard J ; Konycheva, Galina ; Bailey, Graham ; Wheatley, Mark ; Poyner, David R. / Structure-function analysis of RAMP1 by alanine mutagenesis. In: Biochemistry. 2009 ; Vol. 48, No. 1. pp. 198-205.
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