Photoaffinity cross-linking and unnatural amino acid mutagenesis reveal insights into calcitonin gene-related peptide binding to the calcitonin receptor-like receptor/receptor activity-modifying protein 1 (CLR/RAMP1) complex

John Simms, Romez Uddin, Thomas P Sakmar, Joseph J Gingell, Michael L Garelja, Debbie L Hay, Margaret A Brimble, Paul W R Harris, Christopher Arthur Reynolds, David R Poyner

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Abstract

Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). It remains unclear how CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor. In the current study, a photoaffinity cross linker, p-azido L-phenylalanine (azF) was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimised to ensure efficient photolysis of azF residues near to the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of uv-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the 5th transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu12 and Ala13 of CGRP, and Leu16 of CGRP is at the ECL/extracellular domain (ECD) boundary of CLR. Reduced potency, Emax and affinity of [Leu16Ala]-human alpha CGRP is consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation.

Original languageEnglish
Pages (from-to)4915-4922
Number of pages8
JournalBiochemistry
Volume57
Issue number32
Early online date13 Jul 2018
DOIs
Publication statusPublished - 14 Aug 2018

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Receptor Activity-Modifying Protein 1
Calcitonin Receptor-Like Protein
Mutagenesis
Calcitonin Gene-Related Peptide
Amino Acids
Genetic Code
Emitter coupled logic circuits
Molecular Models
Photolysis
Lip
Fluorescein
Phenylalanine

Bibliographical note

This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.

Keywords

  • Journal Article

Cite this

Photoaffinity cross-linking and unnatural amino acid mutagenesis reveal insights into calcitonin gene-related peptide binding to the calcitonin receptor-like receptor/receptor activity-modifying protein 1 (CLR/RAMP1) complex. / Simms, John; Uddin, Romez; Sakmar, Thomas P; Gingell, Joseph J; Garelja, Michael L; Hay, Debbie L; Brimble, Margaret A; Harris, Paul W R; Reynolds, Christopher Arthur; Poyner, David R.

In: Biochemistry, Vol. 57, No. 32, 14.08.2018, p. 4915-4922.

Research output: Contribution to journalArticle

Simms, John ; Uddin, Romez ; Sakmar, Thomas P ; Gingell, Joseph J ; Garelja, Michael L ; Hay, Debbie L ; Brimble, Margaret A ; Harris, Paul W R ; Reynolds, Christopher Arthur ; Poyner, David R. / Photoaffinity cross-linking and unnatural amino acid mutagenesis reveal insights into calcitonin gene-related peptide binding to the calcitonin receptor-like receptor/receptor activity-modifying protein 1 (CLR/RAMP1) complex. In: Biochemistry. 2018 ; Vol. 57, No. 32. pp. 4915-4922.
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T1 - Photoaffinity cross-linking and unnatural amino acid mutagenesis reveal insights into calcitonin gene-related peptide binding to the calcitonin receptor-like receptor/receptor activity-modifying protein 1 (CLR/RAMP1) complex

AU - Simms, John

AU - Uddin, Romez

AU - Sakmar, Thomas P

AU - Gingell, Joseph J

AU - Garelja, Michael L

AU - Hay, Debbie L

AU - Brimble, Margaret A

AU - Harris, Paul W R

AU - Reynolds, Christopher Arthur

AU - Poyner, David R

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PY - 2018/8/14

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N2 - Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). It remains unclear how CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor. In the current study, a photoaffinity cross linker, p-azido L-phenylalanine (azF) was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimised to ensure efficient photolysis of azF residues near to the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of uv-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the 5th transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu12 and Ala13 of CGRP, and Leu16 of CGRP is at the ECL/extracellular domain (ECD) boundary of CLR. Reduced potency, Emax and affinity of [Leu16Ala]-human alpha CGRP is consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation.

AB - Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). It remains unclear how CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor. In the current study, a photoaffinity cross linker, p-azido L-phenylalanine (azF) was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimised to ensure efficient photolysis of azF residues near to the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of uv-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the 5th transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu12 and Ala13 of CGRP, and Leu16 of CGRP is at the ECL/extracellular domain (ECD) boundary of CLR. Reduced potency, Emax and affinity of [Leu16Ala]-human alpha CGRP is consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation.

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JF - Biochemistry

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