Diverse functional motifs within the three intracellular loops of the CGRP1 receptor

Alex C Conner, John Simms, Matthew T Conner, Denise L Wootten, Mark Wheatley, David R Poyner

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

The CGRP(1) receptor exists as a heterodimeric complex between a single-pass transmembrane accessory protein (RAMP1) and a family B G-protein-coupled receptor (GPCR) called the calcitonin receptor-like receptor (CLR). This study investigated the structural motifs found in the intracellular loops (ICLs) of this receptor. Molecular modeling was used to predict active and inactive conformations of each ICL. Conserved residues were altered to alanine by site-directed mutagenesis. cAMP accumulation, cell-surface expression, agonist affinity, and CGRP-stimulated receptor internalization were characterized. Within ICL1, L147 and particularly R151 were important for coupling to G(s). R151 may interact directly with the G-protein, accessing it following conformational changes involving ICL2 and ICL3. At the proximal end of ICL3, I290 and L294, probably lying on the same face of an alpha helix, formed a G-protein coupling motif. The largest effects on coupling were observed with I290A; additionally, it reduced CGRP affinity and impaired internalization. I290 may interact with TM6 to stabilize the conformation of ICL3, but it could also interact directly with Gs. R314, at the distal end of ICL3, impaired G-protein coupling and to a lesser extent reduced CGRP affinity; it may stabilize the TM6-ICL3 junction by interacting with the polar headgroups of membrane phospholipids. Y215 and L214 in ICL2 are required for cell-surface expression; they form a microdomain with H216 which has the same function. This study reveals similarities between the activation of CLR and other GPCRs in the role of TM6 and ICL3 but shows that other conserved motifs differ in their function.

Original languageEnglish
Pages (from-to)12976-12985
Number of pages10
JournalBiochemistry
Volume45
Issue number43
DOIs
Publication statusPublished - 31 Oct 2006

Fingerprint

Calcitonin Receptor-Like Protein
GTP-Binding Proteins
Calcitonin Gene-Related Peptide Receptors
Conformations
Mutagenesis
Amino Acid Motifs
Molecular modeling
Accessories
G-Protein-Coupled Receptors
Site-Directed Mutagenesis
Alanine
Phospholipids
Chemical activation
Membranes
Proteins

Keywords

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Binding Sites
  • COS Cells
  • Cercopithecus aethiops
  • Cyclic AMP
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Receptors, Calcitonin
  • Receptors, Calcitonin Gene-Related Peptide
  • Receptors, G-Protein-Coupled
  • Structure-Activity Relationship
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

Conner, A. C., Simms, J., Conner, M. T., Wootten, D. L., Wheatley, M., & Poyner, D. R. (2006). Diverse functional motifs within the three intracellular loops of the CGRP1 receptor. Biochemistry, 45(43), 12976-12985. https://doi.org/10.1021/bi0615801

Diverse functional motifs within the three intracellular loops of the CGRP1 receptor. / Conner, Alex C; Simms, John; Conner, Matthew T; Wootten, Denise L; Wheatley, Mark; Poyner, David R.

In: Biochemistry, Vol. 45, No. 43, 31.10.2006, p. 12976-12985.

Research output: Contribution to journalArticle

Conner, AC, Simms, J, Conner, MT, Wootten, DL, Wheatley, M & Poyner, DR 2006, 'Diverse functional motifs within the three intracellular loops of the CGRP1 receptor' Biochemistry, vol. 45, no. 43, pp. 12976-12985. https://doi.org/10.1021/bi0615801
Conner AC, Simms J, Conner MT, Wootten DL, Wheatley M, Poyner DR. Diverse functional motifs within the three intracellular loops of the CGRP1 receptor. Biochemistry. 2006 Oct 31;45(43):12976-12985. https://doi.org/10.1021/bi0615801
Conner, Alex C ; Simms, John ; Conner, Matthew T ; Wootten, Denise L ; Wheatley, Mark ; Poyner, David R. / Diverse functional motifs within the three intracellular loops of the CGRP1 receptor. In: Biochemistry. 2006 ; Vol. 45, No. 43. pp. 12976-12985.
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AU - Simms, John

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AU - Wheatley, Mark

AU - Poyner, David R

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AB - The CGRP(1) receptor exists as a heterodimeric complex between a single-pass transmembrane accessory protein (RAMP1) and a family B G-protein-coupled receptor (GPCR) called the calcitonin receptor-like receptor (CLR). This study investigated the structural motifs found in the intracellular loops (ICLs) of this receptor. Molecular modeling was used to predict active and inactive conformations of each ICL. Conserved residues were altered to alanine by site-directed mutagenesis. cAMP accumulation, cell-surface expression, agonist affinity, and CGRP-stimulated receptor internalization were characterized. Within ICL1, L147 and particularly R151 were important for coupling to G(s). R151 may interact directly with the G-protein, accessing it following conformational changes involving ICL2 and ICL3. At the proximal end of ICL3, I290 and L294, probably lying on the same face of an alpha helix, formed a G-protein coupling motif. The largest effects on coupling were observed with I290A; additionally, it reduced CGRP affinity and impaired internalization. I290 may interact with TM6 to stabilize the conformation of ICL3, but it could also interact directly with Gs. R314, at the distal end of ICL3, impaired G-protein coupling and to a lesser extent reduced CGRP affinity; it may stabilize the TM6-ICL3 junction by interacting with the polar headgroups of membrane phospholipids. Y215 and L214 in ICL2 are required for cell-surface expression; they form a microdomain with H216 which has the same function. This study reveals similarities between the activation of CLR and other GPCRs in the role of TM6 and ICL3 but shows that other conserved motifs differ in their function.

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KW - Amino Acid Substitution

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KW - Binding Sites

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KW - Humans

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Protein Binding

KW - Receptors, Calcitonin

KW - Receptors, Calcitonin Gene-Related Peptide

KW - Receptors, G-Protein-Coupled

KW - Structure-Activity Relationship

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

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DO - 10.1021/bi0615801

M3 - Article

VL - 45

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EP - 12985

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

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ER -