Structure-function analysis of RAMP1-RAMP3 chimeras

Tao Qi, John Simms, Richard J. Bailey, Mark Wheatley, Daniel L. Rathbone, Debbie L. Hay, David R Poyner

Research output: Contribution to journalArticle

Abstract

The role of receptor activity modifying protein 1 (RAMP1) in forming receptors with the calcitonin receptor-like receptor (CLR) and the calcitonin receptor (CTR) was examined by producing chimeras between RAMP1 and RAMP3. RAMPs have three extracellular helices. Exchange of helix 1 of the RAMPs or residues 62-69 in helix 2 greatly reduced CLR trafficking (a marker for CLR association). Modeling suggests that these exchanges alter the CLR recognition site on RAMP1, which is more exposed than on RAMP3. Exchange of residues 86-89 of RAMP1 had no effect on the trafficking of CLR but reduced the potency of human (h) alphaCGRP and adrenomedullin. However, these alterations to RAMP1 had no effect on the potency of hbetaCGRP. These residues of RAMP1 lie at the junction of helix 3 and its connecting loop with helix 2. Modeling suggests that the loop is more exposed in RAMP1 than RAMP3; it may play an important role in peptide binding, either directly or indirectly. Exchange of residues 90-94 of RAMP1 caused a modest reduction in CLR expression and a 15-fold decrease in CGRP potency. It is unlikely that the decrease in expression is enough to explain the reduction in potency, and so these may have dual roles in recognizing CLR and CGRP. For CTR, only 6 out of 26 chimeras covering the extracellular part of RAMP1 did not reduce agonist potency. Thus the association of CTR with RAMP1 seems more sensitive to changes in RAMP1 structure induced by the chimeras than is CLR.
LanguageEnglish
Pages522-531
Number of pages10
JournalBiochemistry
Volume49
Issue number3
DOIs
Publication statusPublished - 17 Dec 2010

Fingerprint

Receptor Activity-Modifying Protein 1
Calcitonin Receptor-Like Protein
Calcitonin Receptors
Association reactions
Adrenomedullin

Keywords

  • adrenomedullin
  • amino acid sequence
  • animals
  • binding sites
  • COS cells
  • calcitonin receptor-like protein
  • cell line
  • cercopithecus aethiops
  • conserved sequence
  • humans
  • intracellular signaling peptides and proteins
  • membrane proteins
  • molecular sequence data
  • rats
  • receptor activity-modifying protein 1
  • receptor activity-modifying protein 3
  • receptor activity-modifying proteins
  • calcitonin receptors
  • recombinant fusion proteins
  • structure-activity relationship
  • transfection

Cite this

Qi, Tao ; Simms, John ; Bailey, Richard J. ; Wheatley, Mark ; L. Rathbone, Daniel ; Hay, Debbie L. ; Poyner, David R. / Structure-function analysis of RAMP1-RAMP3 chimeras. In: Biochemistry. 2010 ; Vol. 49, No. 3. pp. 522-531.
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abstract = "The role of receptor activity modifying protein 1 (RAMP1) in forming receptors with the calcitonin receptor-like receptor (CLR) and the calcitonin receptor (CTR) was examined by producing chimeras between RAMP1 and RAMP3. RAMPs have three extracellular helices. Exchange of helix 1 of the RAMPs or residues 62-69 in helix 2 greatly reduced CLR trafficking (a marker for CLR association). Modeling suggests that these exchanges alter the CLR recognition site on RAMP1, which is more exposed than on RAMP3. Exchange of residues 86-89 of RAMP1 had no effect on the trafficking of CLR but reduced the potency of human (h) alphaCGRP and adrenomedullin. However, these alterations to RAMP1 had no effect on the potency of hbetaCGRP. These residues of RAMP1 lie at the junction of helix 3 and its connecting loop with helix 2. Modeling suggests that the loop is more exposed in RAMP1 than RAMP3; it may play an important role in peptide binding, either directly or indirectly. Exchange of residues 90-94 of RAMP1 caused a modest reduction in CLR expression and a 15-fold decrease in CGRP potency. It is unlikely that the decrease in expression is enough to explain the reduction in potency, and so these may have dual roles in recognizing CLR and CGRP. For CTR, only 6 out of 26 chimeras covering the extracellular part of RAMP1 did not reduce agonist potency. Thus the association of CTR with RAMP1 seems more sensitive to changes in RAMP1 structure induced by the chimeras than is CLR.",
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Structure-function analysis of RAMP1-RAMP3 chimeras. / Qi, Tao; Simms, John; Bailey, Richard J.; Wheatley, Mark; L. Rathbone, Daniel; Hay, Debbie L.; Poyner, David R.

In: Biochemistry, Vol. 49, No. 3, 17.12.2010, p. 522-531.

Research output: Contribution to journalArticle

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T1 - Structure-function analysis of RAMP1-RAMP3 chimeras

AU - Qi, Tao

AU - Simms, John

AU - Bailey, Richard J.

AU - Wheatley, Mark

AU - L. Rathbone, Daniel

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AU - Poyner, David R

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AB - The role of receptor activity modifying protein 1 (RAMP1) in forming receptors with the calcitonin receptor-like receptor (CLR) and the calcitonin receptor (CTR) was examined by producing chimeras between RAMP1 and RAMP3. RAMPs have three extracellular helices. Exchange of helix 1 of the RAMPs or residues 62-69 in helix 2 greatly reduced CLR trafficking (a marker for CLR association). Modeling suggests that these exchanges alter the CLR recognition site on RAMP1, which is more exposed than on RAMP3. Exchange of residues 86-89 of RAMP1 had no effect on the trafficking of CLR but reduced the potency of human (h) alphaCGRP and adrenomedullin. However, these alterations to RAMP1 had no effect on the potency of hbetaCGRP. These residues of RAMP1 lie at the junction of helix 3 and its connecting loop with helix 2. Modeling suggests that the loop is more exposed in RAMP1 than RAMP3; it may play an important role in peptide binding, either directly or indirectly. Exchange of residues 90-94 of RAMP1 caused a modest reduction in CLR expression and a 15-fold decrease in CGRP potency. It is unlikely that the decrease in expression is enough to explain the reduction in potency, and so these may have dual roles in recognizing CLR and CGRP. For CTR, only 6 out of 26 chimeras covering the extracellular part of RAMP1 did not reduce agonist potency. Thus the association of CTR with RAMP1 seems more sensitive to changes in RAMP1 structure induced by the chimeras than is CLR.

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KW - amino acid sequence

KW - animals

KW - binding sites

KW - COS cells

KW - calcitonin receptor-like protein

KW - cell line

KW - cercopithecus aethiops

KW - conserved sequence

KW - humans

KW - intracellular signaling peptides and proteins

KW - membrane proteins

KW - molecular sequence data

KW - rats

KW - receptor activity-modifying protein 1

KW - receptor activity-modifying protein 3

KW - receptor activity-modifying proteins

KW - calcitonin receptors

KW - recombinant fusion proteins

KW - structure-activity relationship

KW - transfection

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SP - 522

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T2 - Biochemistry

JF - Biochemistry

SN - 0006-2960

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