Characterization of Antibodies against Receptor Activity-Modifying Protein 1 (RAMP1): A Cautionary Tale

Calcitonin gene-related peptide (CGRP) is a key component of migraine pathophysiology, yielding effective migraine therapeutics. CGRP receptors contain a core accessory protein subunit: receptor activity-modifying protein 1 (RAMP1). Understanding of RAMP1 expression is incomplete, partly due to the challenges in identifying specific and validated antibody tools. We profiled antibodies for immunodetection of RAMP1 using Western blotting, immunocytochemistry and immunohistochemistry, including using RAMP1 knockout mouse tissue. Most antibodies could detect RAMP1 in Western blotting and immunocytochemistry using transfected cells. Two antibodies (844, ab256575) could detect a RAMP1-like band in Western blots of rodent brain but not RAMP1 knockout mice. However, cross-reactivity with other proteins was evident for all antibodies. This cross-reactivity prevented clear conclusions about RAMP1 anatomical localization, as each antibody detected a distinct pattern of immunoreactivity in rodent brain. We cannot confidently attribute immunoreactivity produced by RAMP1 antibodies (including 844) to the presence of RAMP1 protein in immunohistochemical applications in brain tissue. RAMP1 expression in brain and other tissues therefore needs to be revisited using RAMP1 antibodies that have been comprehensively validated using multiple strategies to establish multiple lines of convincing evidence. As RAMP1 is important for other GPCR/ligand pairings, our results have broader significance beyond the CGRP field

Distinct Patterns of Internalization of Different Calcitonin GeneRelated Peptide Receptors

Calcitonin gene-related peptide (CGRP) is a neuropeptide that is involved in the transmission of pain. Drugs targeting CGRP or a CGRP receptor are efficacious in the treatment of migraine. The canonical CGRP receptor is a complex of a G protein-coupled receptor, the calcitonin-like receptor (CLR), with an accessory protein, receptor activity-modifying protein 1 (RAMP1). A second receptor, the AMY1 receptor, a complex of the calcitonin receptor with RAMP1, is a dual high-affinity receptor for CGRP and amylin. Receptor regulatory processes, such as internalization, are crucial for controlling peptide and drug responsiveness. Given the importance of CGRP receptor activity in migraine we compared the internalization profiles of both receptors for CGRP using novel fluorescent probes and a combination of live cell imaging, fixed cell imaging, and ELISA. This revealed stark differences in the regulation of each receptor with the AMY1 receptor unexpectedly showing little internalization.Peer Reviewe

Calcitonin receptor antibody validation and expression in the rodent brain

Background and aim Therapeutics that reduce calcitonin gene-related peptide activity are effective migraine treatments. However, gaps remain in our understanding of the molecular mechanisms that link calcitonin gene-related peptide to migraine. The amylin 1 receptor responds potently to calcitonin gene-related peptide, and to the related peptide amylin, but its role in relation to either peptide or to migraine is unclear. We sought to better understand the expression of the amylin 1 receptor protein subunit, the calcitonin receptor, in the rodent brain. Methods We profiled three antibodies for immunodetection of calcitonin receptor, using immunocytochemistry, western blotting, and calcitonin receptor conditional knockout mouse tissue. Selected migraine-relevant rat brain regions were then examined for calcitonin receptor-like immunoreactivity. Results All three antibodies detected calcitonin receptor protein but only one (188/10) produced robust immunostaining in rodent brain, under the conditions used. Calcitonin receptor-like immunoreactivity was apparent in the rat brainstem and midbrain including the locus coeruleus, periaqueductal grey and spinal trigeminal nucleus. Conclusions Anti-calcitonin receptor antibodies require comprehensive profiling to ensure confidence in the detection of calcitonin receptor. Using a validated antibody, calcitonin receptor-like immunoreactivity was detected in several brain regions relevant to migraine. Further research is needed to understand the functional consequences of calcitonin receptor expression for calcitonin gene-related peptide or amylin physiology and pathophysiology

Characterization of Antibodies against Receptor Activity-Modifying Protein 1 (RAMP1): A Cautionary Tale

Calcitonin gene-related peptide (CGRP) is a key component of migraine pathophysiology, yielding effective migraine therapeutics. CGRP receptors contain a core accessory protein subunit: receptor activity-modifying protein 1 (RAMP1). Understanding of RAMP1 expression is incomplete, partly due to the challenges in identifying specific and validated antibody tools. We profiled antibodies for immunodetection of RAMP1 using Western blotting, immunocytochemistry and immunohistochemistry, including using RAMP1 knockout mouse tissue. Most antibodies could detect RAMP1 in Western blotting and immunocytochemistry using transfected cells. Two antibodies (844, ab256575) could detect a RAMP1-like band in Western blots of rodent brain but not RAMP1 knockout mice. However, cross-reactivity with other proteins was evident for all antibodies. This cross-reactivity prevented clear conclusions about RAMP1 anatomical localization, as each antibody detected a distinct pattern of immunoreactivity in rodent brain. We cannot confidently attribute immunoreactivity produced by RAMP1 antibodies (including 844) to the presence of RAMP1 protein in immunohistochemical applications in brain tissue. RAMP1 expression in brain and other tissues therefore needs to be revisited using RAMP1 antibodies that have been comprehensively validated using multiple strategies to establish multiple lines of convincing evidence. As RAMP1 is important for other GPCR/ligand pairings, our results have broader significance beyond the CGRP field

Molecular Signature for Receptor Engagement in the Metabolic Peptide Hormone Amylin

The pancreatic peptide hormone, amylin, plays a critical role in the control of appetite, and synergizes with other key metabolic hormones such as glucagon-like peptide 1 (GLP-1). There is opportunity to develop potent and long-acting analogues of amylin or hybrids between these and GLP-1 mimetics for treating obesity. To achieve this, interrogation of how the 37 amino acid amylin peptide engages with its complex receptor system is required. We synthesized an extensive library of peptides to profile the human amylin sequence, determining the role of its disulfide loop, amidated <i>C</i>-terminus and receptor “capture” and “activation” regions in receptor signaling. We profiled four signaling pathways with different ligands at multiple receptor subtypes, in addition to exploring selectivity determinants between related receptors. Distinct roles for peptide subregions in receptor binding and activation were identified, resulting in peptides with greater activity than the native sequence. Enhanced peptide activity was preserved in the brainstem, the major biological target for amylin. Interpretation of our data using full-length active receptor models supported by molecular dynamics, metadynamics, and supervised molecular dynamics simulations guided the synthesis of a potent dual agonist of GLP-1 and amylin receptors. The data offer new insights into the function of peptide amidation, how allostery drives peptide–receptor interactions, and provide a valuable resource for the development of novel amylin agonists for treating diabetes and obesity

Molecular Signature for Receptor Engagement in the Metabolic Peptide Hormone Amylin

The pancreatic peptide hormone, amylin, plays a critical role in the control of appetite, and synergizes with other key metabolic hormones such as glucagon-like peptide 1 (GLP-1). There is opportunity to develop potent and long-acting analogues of amylin or hybrids between these and GLP-1 mimetics for treating obesity. To achieve this, interrogation of how the 37 amino acid amylin peptide engages with its complex receptor system is required. We synthesized an extensive library of peptides to profile the human amylin sequence, determining the role of its disulfide loop, amidated <i>C</i>-terminus and receptor “capture” and “activation” regions in receptor signaling. We profiled four signaling pathways with different ligands at multiple receptor subtypes, in addition to exploring selectivity determinants between related receptors. Distinct roles for peptide subregions in receptor binding and activation were identified, resulting in peptides with greater activity than the native sequence. Enhanced peptide activity was preserved in the brainstem, the major biological target for amylin. Interpretation of our data using full-length active receptor models supported by molecular dynamics, metadynamics, and supervised molecular dynamics simulations guided the synthesis of a potent dual agonist of GLP-1 and amylin receptors. The data offer new insights into the function of peptide amidation, how allostery drives peptide–receptor interactions, and provide a valuable resource for the development of novel amylin agonists for treating diabetes and obesity