Selective CGRP and adrenomedullin peptide binding by tethered RAMP-calcitonin receptor-like receptor extracellular domain fusion proteins

Protein Sci. 2013 Dec;22(12):1775-85. doi: 10.1002/pro.2377. Epub 2013 Oct 19.

Abstract

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are related peptides that are potent vasodilators. The CGRP and AM receptors are heteromeric protein complexes comprised of a shared calcitonin receptor-like receptor (CLR) subunit and a variable receptor activity modifying protein (RAMP) subunit. RAMP1 enables CGRP binding whereas RAMP2 confers AM specificity. How RAMPs determine peptide selectivity is unclear and the receptor stoichiometries are a topic of debate with evidence for 1:1, 2:2, and 2:1 CLR:RAMP stoichiometries. Here, we describe bacterial production of recombinant tethered RAMP-CLR extracellular domain (ECD) fusion proteins and biochemical characterization of their peptide binding properties. Tethering the two ECDs ensures complex stability and enforces defined stoichiometry. The RAMP1-CLR ECD fusion purified as a monomer, whereas the RAMP2-CLR ECD fusion purified as a dimer. Both proteins selectively bound their respective peptides with affinities in the low micromolar range. Truncated CGRP(27-37) and AM(37-52) fragments were identified as the minimal ECD complex binding regions. The CGRP C-terminal amide group contributed to, but was not required for, ECD binding, whereas the AM C-terminal amide group was essential for ECD binding. Alanine-scan experiments identified CGRP residues T30, V32, and F37 and AM residues P43, K46, I47, and Y52 as critical for ECD binding. Our results identify CGRP and AM determinants for receptor ECD complex binding and suggest that the CGRP receptor functions as a 1:1 heterodimer. In contrast, the AM receptor may function as a 2:2 dimer of heterodimers, although our results cannot rule out 2:1 or 1:1 stoichiometries.

Keywords: calcitonin family peptide; class B G protein-coupled receptor; neuropeptide; vasodilator.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adrenomedullin / chemistry
  • Adrenomedullin / metabolism*
  • Amino Acid Sequence
  • Binding Sites
  • Calcitonin Gene-Related Peptide / chemistry
  • Calcitonin Gene-Related Peptide / metabolism*
  • Calcitonin Receptor-Like Protein / chemistry
  • Calcitonin Receptor-Like Protein / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Humans
  • Peptides / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Receptor Activity-Modifying Proteins / metabolism*
  • Receptors, Adrenomedullin / chemistry
  • Receptors, Adrenomedullin / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction

Substances

  • Calcitonin Receptor-Like Protein
  • Peptides
  • Receptor Activity-Modifying Proteins
  • Receptors, Adrenomedullin
  • Recombinant Fusion Proteins
  • Adrenomedullin
  • Calcitonin Gene-Related Peptide