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.
LanguageEnglish
Pages8434-8444
Number of pages11
JournalBiochemistry
Volume47
Issue number32
DOIs
Publication statusPublished - 18 Jul 2008

Fingerprint

Calcitonin Gene-Related Peptide Receptors
G-Protein-Coupled Receptors
Membranes
Sequence Homology
Circular Dichroism
Liposomes
Tryptophan
Nuclear magnetic resonance spectroscopy
Magnetic Resonance Spectroscopy
Fluorescence
Peptides
Mutation

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

Cite this

Conner, M., Hicks, M. R., Dafforn, T., Knowles, T. J., Ludwig, C., Staddon, S., ... 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
Conner, Matthew ; Hicks, Matthew R. ; Dafforn, Tim ; Knowles, Timothy J. ; Ludwig, Christian ; Staddon, Susan ; Overduin, Michael ; Günther, Ulrich L. ; Thome, Johannes ; Wheatley, M. ; Poyner, David R. ; Conner, Alex C. / Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR. In: Biochemistry. 2008 ; Vol. 47, No. 32. pp. 8434-8444.
@article{19b68fb6e79b4812b9a726580ec1f243,
title = "Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR",
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.",
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",
author = "Matthew Conner and Hicks, {Matthew R.} and Tim Dafforn and Knowles, {Timothy J.} and Christian Ludwig and Susan Staddon and Michael Overduin and G{\"u}nther, {Ulrich L.} and Johannes Thome and M. Wheatley and Poyner, {David R.} and Conner, {Alex C.}",
year = "2008",
month = "7",
day = "18",
doi = "10.1021/bi8004126",
language = "English",
volume = "47",
pages = "8434--8444",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "32",

}

Conner, M, Hicks, MR, Dafforn, T, Knowles, TJ, Ludwig, C, Staddon, S, Overduin, M, Günther, UL, Thome, J, Wheatley, M, Poyner, DR & Conner, AC 2008, 'Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR' Biochemistry, vol. 47, no. 32, pp. 8434-8444. https://doi.org/10.1021/bi8004126

Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR. / Conner, Matthew; Hicks, Matthew R.; Dafforn, Tim; Knowles, Timothy J.; Ludwig, Christian; Staddon, Susan; Overduin, Michael; Günther, Ulrich L.; Thome, Johannes; Wheatley, M.; Poyner, David R.; Conner, Alex C.

In: Biochemistry, Vol. 47, No. 32, 18.07.2008, p. 8434-8444.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR

AU - Conner, Matthew

AU - Hicks, Matthew R.

AU - Dafforn, Tim

AU - Knowles, Timothy J.

AU - Ludwig, Christian

AU - Staddon, Susan

AU - Overduin, Michael

AU - Günther, Ulrich L.

AU - Thome, Johannes

AU - Wheatley, M.

AU - Poyner, David R.

AU - Conner, Alex C.

PY - 2008/7/18

Y1 - 2008/7/18

N2 - 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.

AB - 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.

KW - G-protein coupled receptors

KW - GPCRs

KW - C-terminus

KW - membrane-parallel

KW - eighth helix

KW - mutations

KW - N400

KW - C436

KW - CGRP-mediated internalization

KW - G388

KW - W399)

KW - receptor localization

KW - cell surface

KW - dichroism

KW - waterLOGSY

KW - NMR spectroscopy

KW - SALMON

KW - peptide

KW - interfacial tryptophan residue

UR - http://www.scopus.com/inward/record.url?scp=49449095044&partnerID=8YFLogxK

UR - http://pubs.acs.org/doi/full/10.1021/bi8004126

U2 - 10.1021/bi8004126

DO - 10.1021/bi8004126

M3 - Article

VL - 47

SP - 8434

EP - 8444

JO - Biochemistry

T2 - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 32

ER -

Conner M, Hicks MR, Dafforn T, Knowles TJ, Ludwig C, Staddon S et al. Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR. Biochemistry. 2008 Jul 18;47(32):8434-8444. https://doi.org/10.1021/bi8004126