Structure−Function Analysis of RAMP1 by Alanine Mutagenesis

Simms, John; Hay, Debbie L.; Bailey, Richard J.; Konycheva, Galina; Bailey, Graham S.; Wheatley, Mark; Poyner, David R.

doi:10.1021/bi801869n

Cited by 22 publications

(26 citation statements)

References 22 publications

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“…Although mouse Ramp1 has three isoforms, including an isoform that utilizes an alternate exon 3 from the form encoded by isoform 1, three-dimensional structure prediction models of isoforms 2 and 3 indicate that these variants lack the third α–helix and have an incomplete second α–helix. Additionally, isoforms 2 and 3 lack the critical residues described by Simms et al as necessary for CGRP interaction and cAMP generation [25], indicating that it is unlikely that these isoforms can interact with CLR to form a functional CGRP receptor. Using standard molecular biology techniques, we flanked the third exon of the mouse Ramp1 gene with loxP sites to generate the targeting vector ( Figure 1A ).…”

Section: Resultsmentioning

confidence: 93%

“…Since CGRP can signal via the Ramp1/Calcrl receptor, we were interested in determining whether a loss Ramp1 could also result in an attenuation of airway hyperresponsiveness after sensitization to ovalbumin and a subsequent challenge period to reflect the findings of Aoki-Nagase et al Therefore, we developed a global Ramp1 knockout mouse model, referred to as Ramp1 −/− mice for the duration of this article. Studies performed by other groups have demonstrated that the second and third extracellular α–helices of Ramp1 contain residues critical for CGRP binding and subsequent cAMP generation [25]. Therefore, we decided to excise exon 3 of isoform 1 of the mouse Ramp1 gene, which codes for these extracellular loops, to remove these critical residues and prevent the formation of a functional CGRP receptor.…”

Section: Resultsmentioning

confidence: 99%

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Deficiency of RAMP1 Attenuates Antigen-Induced Airway Hyperresponsiveness in Mice

Wetzel-Strong

Hua

et al. 2014

PLoS ONE

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Asthma is a chronic inflammatory disease affecting the lung, characterized by breathing difficulty during an attack following exposure to an environmental trigger. Calcitonin gene-related peptide (CGRP) is a neuropeptide that may have a pathological role in asthma. The CGRP receptor is comprised of two components, which include the G-protein coupled receptor, calcitonin receptor-like receptor (CLR), and receptor activity-modifying protein 1 (RAMP1). RAMPs, including RAMP1, mediate ligand specificity in addition to aiding in the localization of receptors to the cell surface. Since there has been some controversy regarding the effect of CGRP on asthma, we sought to determine the effect of CGRP signaling ablation in an animal model of asthma. Using gene-targeting techniques, we generated mice deficient for RAMP1 by excising exon 3. After determining that these mice are viable and overtly normal, we sensitized the animals to ovalbumin prior to assessing airway resistance and inflammation after methacholine challenge. We found that mice lacking RAMP1 had reduced airway resistance and inflammation compared to wildtype animals. Additionally, we found that a 50% reduction of CLR, the G-protein receptor component of the CGRP receptor, also ameliorated airway resistance and inflammation in this model of allergic asthma. Interestingly, the loss of CLR from the smooth muscle cells did not alter the airway resistance, indicating that CGRP does not act directly on the smooth muscle cells to drive airway hyperresponsiveness. Together, these data indicate that signaling through RAMP1 and CLR plays a role in mediating asthma pathology. Since RAMP1 and CLR interact to form a receptor for CGRP, our data indicate that aberrant CGRP signaling, perhaps on lung endothelial and inflammatory cells, contributes to asthma pathophysiology. Finally, since RAMP-receptor interfaces are pharmacologically tractable, it may be possible to develop compounds targeting the RAMP1/CLR interface to assist in the treatment of asthma.

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Section: Resultsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Deficiency of RAMP1 Attenuates Antigen-Induced Airway Hyperresponsiveness in Mice

Wetzel-Strong

Hua

et al. 2014

PLoS ONE

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“…Stimulation with agonists and assay of cAMP was by a radioreceptor assay as described elsewhere [27].…”

Section: Methodsmentioning

confidence: 99%

Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function

Barwell

Conner

Poyner

2011

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced αCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced αCGRP binding. These residues form a hydrophobic cluster within an area defined as the “minor groove” of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of αCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on αCGRP binding and cAMP production; they are likely to indirectly influence the binding site for αCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired αCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.

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“…Stimulation with agonists and assay of cAMP was by a radioreceptor assay as described elsewhere [28].…”

Section: Methodsmentioning

confidence: 99%

“…A negative control of myc RAMP1/empty pcDNA3.1(−) was used. The ELISA was carried out as described previously to measure both cell surface and total expression of CLR [28]. Cell surface expression data was normalized to make mean WT expression 100% and the mean myc RAMP1/empty pcDNA3.1(−) vector as 0%.…”

Section: Methodsmentioning

confidence: 99%

Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23–60 of the calcitonin receptor-like receptor

Barwell¹,

Miller²,

Donnelly³

et al. 2010

Peptides

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The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs.

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Structure−Function Analysis of RAMP1 by Alanine Mutagenesis

Cited by 22 publications

References 22 publications

Deficiency of RAMP1 Attenuates Antigen-Induced Airway Hyperresponsiveness in Mice

Deficiency of RAMP1 Attenuates Antigen-Induced Airway Hyperresponsiveness in Mice

Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function

Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23–60 of the calcitonin receptor-like receptor

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