GPCRs globally coevolved with receptor activity-modifying proteins, RAMPs

Barbash, Shahar; Lorenzen, Emily; Persson, Torbjörn; Huber, Thomas; Sakmar, Thomas P.

doi:10.1073/pnas.1713074114

Cited by 32 publications

(30 citation statements)

References 22 publications

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“…All of the secretin-like receptors were included in the test set because this GPCR family encompasses the majority of GPCR-RAMP interactions described to date. Based on an earlier co-evolution and co-expression analyses we also included GPR4 and GPR182 from the rhodopsin-like GPCR family, and ADGRF5 from the adhesion GPCR family (14) . In addition, the chemokine cluster of GPCRs demonstrated significantly higher median co-expression with RAMP2 and RAMP3 in comparison with all GPCRs (fig.…”

Section: Resultsmentioning

confidence: 99%

“…Several lines of evidence from disease-associated pathologies, tissue expression and knock out mice indicate that the breadth of GPCR-RAMP interactions is not yet fully appreciated (12, 13) . Furthermore, we demonstrated recently that GPCRs and RAMPs globally co-evolved and display correlated mRNA levels across human tissues, which suggests the likelihood for more widespread GPCR-RAMP interactions (14) . We also showed concordance between mRNA levels for a subset of GPCRs and RAMP2 using a single-cell MERFISH (multiplexed error reduced fluorescence in situ hybridization) method (15) .…”

Section: Introductionmentioning

confidence: 94%

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Multiplexed Analysis of the Secretin-like GPCR-RAMP Interactome

Lorenzen

Dodig‐Crnković

Kotliar

et al. 2019

Preprint

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20Although receptor activity-modifying proteins (RAMPs) have been shown to modulate the 21 functions of several different G protein-coupled receptors (GPCRs), potential direct interactions 22 among the three known RAMPs and hundreds of GPCRs has never been investigated. We 23 engineered three epitope-tagged RAMPs and 23 epitope-tagged GPCRs, focusing on the secretin-24 like family of GPCRs, and developed a suspension bead array (SBA) immunoassay designed to 25 detect RAMP-GPCR complexes. We then used 64 antibodies raised against native RAMPs and 26 GPCRs, along with four antibodies targeting the epitope tags, to multiplex the SBA assay to 27 detect and measure all possible combinations of interaction among the 23 GPCRs and three 28 RAMPs. The results of the SBA assay provide a complete interactome of secretin-like GPCRs 29 with RAMPs. We demonstrate direct interaction of previously reported secretin-like GPCRs 30 whose functions are modulated by RAMPs. We also discovered novel sets of GPCR-RAMP 31 interacting pairs, and found additional secretin-like GPCRs, chemokine receptors and orphan 32 receptors that interact with RAMPs. Using in situ proximity ligation assay, we verified a subset of 33 these novel GPCR-RAMP interactions in cell membranes. In total, we found GPCR-RAMP 34 interactions for the majority of the 23 GPCRs tested. Each GPCR interacted with either all three 35 RAMPs or with RAMP2 and RAMP3, with the exception of one GPCR that interacted with just 36 RAMP3. In summary, we describe an SBA strategy that will be useful to search for GPCR-37 RAMP interactions in cell lines and tissues, and conclude that GPCR-RAMP interactions are 38 more common than previously appreciated. 39 40 42 commonly targeted for drug development. Emerging evidence suggests that the surface expression 43 and activity of GPCRs can be modulated by receptor activity-modifying proteins (RAMPs), a 44 ubiquitously-expressed family of single-pass membrane proteins with only three members. First discovered as a necessary component for the function of calcitonin-like receptor (CALCRL), 46 RAMPs were shown to alter ligand specificity of both CALCRL and calcitonin receptor (CALCR) 47 by dictating to which ligand they respond , (1-3). Interaction of RAMPs with GPCRs has since been 48 demonstrated with several additional members of the secretin-like GPCRs. RAMPs can influence 49 several features of GPCR biology, including trafficking to the cell membrane, ligand specificity, 50 downstream signaling and recycling (4-11). However, many of the secretin-like GPCR-RAMP 51 interactions remain unverified, and most of the interactions have not been demonstrated using a 52 direct binding assay. In addition, prior reports show conflicting results regarding whether certain 53 secretin-like GPCRs form complexes with RAMPs. 54There are also suggestions that RAMPs form complexes with a broader set of GPCRs. A 55 single GPCR from the glutamate-like family, calcium-sensing receptor (CaSR), and a single GPCR 56 from the rhodopsin-like family, G protein-coup...

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 94%

Multiplexed Analysis of the Secretin-like GPCR-RAMP Interactome

Lorenzen

Dodig‐Crnković

Kotliar

et al. 2019

Preprint

Self Cite

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“…However, these pleiotropic effects of RAMPs have mostly been studied in the context of a few receptors in the Family B/Secretin family, including the calcitonin receptor (CTR), secretin receptor, glucagon (6, 7), CRF 1 (8,9), and VPAC 1/2 (8) and the Family A receptors CaSR (10) and GPR30/GPER1 (11). The strong coevolution of RAMPs with most GPCR families suggests that they may have expanded interacting partners (12,13).…”

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confidence: 99%

RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis

Mackie

Nielsen

Harris

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Receptor-activity–modifying proteins (RAMPs) are single transmembrane-spanning proteins which serve as molecular chaperones and allosteric modulators of G-protein–coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have been previously studied in the context of their effects on Family B GPCRs, the coevolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions. Using bioluminescence resonance energy transfer-based and cell-surface expression approaches, we comprehensively screen for RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and nearly all chemokine receptors. Most notably, we identify robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cell migration. Specifically, RAMP3 association with atypical chemokine receptor 3 (ACKR3) diminishes adrenomedullin (AM) ligand availability without changing G-protein coupling. Instead, RAMP3 is required for the rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following either AM or SDF-1/CXCL12 binding, thereby enabling formation of dynamic spatiotemporal chemotactic gradients. Consequently, genetic deletion of either ACKR3 or RAMP3 in mice abolishes directed cell migration of retinal angiogenesis. Thus, RAMP association with chemokine receptor family members represents a molecular interaction to control receptor signaling and trafficking properties.

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“…with several other GPCRs (18), and a recent bioinformatics analysis suggested that RAMPs interact with more GPCRs than is currently appreciated (22). Understanding how the three peptides bind the receptors is especially important considering the phenomenon of signaling bias in which different ligands stabilize distinct receptor conformations to yield different downstream signaling outcomes (23).…”

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confidence: 99%

Structure–function analyses reveal a triple β-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design

Roehrkasse

Booe

Lee

et al. 2018

Journal of Biological Chemistry

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The cardioprotective vasodilator peptide adrenomedullin 2/intermedin (AM2/IMD) and the related adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) signal through three heterodimeric receptors comprising the calcitonin receptor-like class B G protein-coupled receptor (CLR) and a variable receptor activity-modifying protein (RAMP1, -2, or -3) that determines ligand selectivity. The CGRP receptor (RAMP1:CLR) favors CGRP binding, whereas the AM (RAMP2:CLR) and AM (RAMP3:CLR) receptors favor AM binding. How AM2/IMD binds the receptors and how RAMPs modulate its binding is unknown. Here, we show that AM2/IMD binds the three purified RAMP-CLR extracellular domain (ECD) complexes with a selectivity profile that is distinct from those of CGRP and AM. AM2/IMD bound all three ECD complexes but preferred the CGRP and AM receptor complexes. A 2.05 Å resolution crystal structure of an AM2/IMD antagonist fragment-bound RAMP1-CLR ECD complex revealed that AM2/IMD binds the complex through a unique triple β-turn conformation that was confirmed by peptide and receptor mutagenesis. Comparisons of the receptor-bound conformations of AM2/IMD, AM, and a high-affinity CGRP analog revealed differences that may have implications for biased signaling. Guided by the structure, enhanced-affinity AM2/IMD antagonist variants were developed, including one that discriminates the AM and AM receptors with ∼40-fold difference in affinities and one stabilized by an intramolecular disulfide bond. These results reveal differences in how the three peptides engage the receptors, inform development of AM2/IMD-based pharmacological tools and therapeutics, and provide insights into RAMP modulation of receptor pharmacology.

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GPCRs globally coevolved with receptor activity-modifying proteins, RAMPs

Cited by 32 publications

References 22 publications

Multiplexed Analysis of the Secretin-like GPCR-RAMP Interactome

Multiplexed Analysis of the Secretin-like GPCR-RAMP Interactome

RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis

Structure–function analyses reveal a triple β-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design

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