Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR

Matthew Conner, Matthew R. Hicks, Tim Dafforn, Timothy J. Knowles, Christian Ludwig, Susan Staddon, Michael Overduin, Ulrich L. Günther, Johannes Thome, M. Wheatley, David R. Poyner, Alex C. Conner

Research output: Contribution to journalArticle

Abstract

G-protein coupled receptors (GPCRs) typically have a functionally important C-terminus which, in the largest subfamily (family A), includes a membrane-parallel eighth helix. Mutations of this region are associated with several diseases. There are few C-terminal studies on the family B GPCRs and no data supporting the existence of a similar eighth helix in this second major subfamily, which has little or no sequence homology to family A GPCRs. Here we show that the C-terminus of a family B GPCR (CLR) has a disparate region from N400 to C436 required for CGRP-mediated internalization, and a proximal region of twelve residues (from G388 to W399), in a similar position to the family A eighth helix, required for receptor localization at the cell surface. A combination of circular and linear dichroism, fluorescence and modified waterLOGSY NMR spectroscopy (SALMON) demonstrated that a peptide mimetic of this domain readily forms a membrane-parallel helix anchored to the liposome by an interfacial tryptophan residue. The study reveals two key functions held within the C-terminus of a family B GPCR and presents support for an eighth helical region with striking topological similarity to the nonhomologous family A receptor. This helix structure appears to be found in most other family B GPCRs.
Original languageEnglish
Pages (from-to)8434-8444
Number of pages11
JournalBiochemistry
Volume47
Issue number32
DOIs
Publication statusPublished - 18 Jul 2008

Keywords

  • G-protein coupled receptors
  • GPCRs
  • C-terminus
  • membrane-parallel
  • eighth helix
  • mutations
  • N400
  • C436
  • CGRP-mediated internalization
  • G388
  • W399)
  • receptor localization
  • cell surface
  • dichroism
  • waterLOGSY
  • NMR spectroscopy
  • SALMON
  • peptide
  • interfacial tryptophan residue

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    Conner, M., Hicks, M. R., Dafforn, T., Knowles, T. J., Ludwig, C., Staddon, S., Overduin, M., Günther, U. L., Thome, J., Wheatley, M., Poyner, D. R., & Conner, A. C. (2008). Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR. Biochemistry, 47(32), 8434-8444. https://doi.org/10.1021/bi8004126