The effects of RAMPs upon cell signalling

Routledge, Sarah J; Ladds, Graham; Poyner, David R.

doi:10.1016/j.mce.2017.03.033

Cited by 15 publications

(12 citation statements)

References 64 publications

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“…The method that we present detects interactions occurring anywhere in the cells, as opposed to just the cell membrane. Of note, preliminary data from another report did suggest that RAMPs were detected at the cell surface when coexpressed with GLP2R in HEK293T cells (25).…”

Section: Discussionmentioning

confidence: 97%

Multiplexed analysis of the secretin-like GPCR-RAMP interactome

et al. 2019

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Receptor activity–modifying proteins (RAMPs) have been shown to modulate the functions of several G protein–coupled receptors (GPCRs), but potential direct interactions among the three known RAMPs and hundreds of GPCRs have never been investigated. Focusing mainly on the secretin-like family of GPCRs, we engineered epitope-tagged GPCRs and RAMPs, and developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized lysates. Using 64 antibodies raised against the native proteins and 4 antibodies targeting the epitope tags, we mapped the interactions among 23 GPCRs and 3 RAMPs. We validated nearly all previously reported secretin-like GPCR-RAMP interactions, and also found previously unidentified RAMP interactions with additional secretin-like GPCRs, chemokine receptors, and orphan receptors. The results provide a complete interactome of secretin-like GPCRs with RAMPs. The SBA strategy will be useful to search for additional GPCR-RAMP complexes and other interacting membrane protein pairs in cell lines and tissues.

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Section: Discussionmentioning

confidence: 97%

Multiplexed analysis of the secretin-like GPCR-RAMP interactome

et al. 2019

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“…The method we present detects interactions occurring anywhere in the cells, as opposed to just the cell membrane. Of note, preliminary data from another report did suggest that RAMPs were detected at the cell surface when co-expressed with GLP2R in HEK293T cells (24) .…”

Section: Discussionmentioning

confidence: 99%

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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“…RAMPs have been shown to interact with a wide range of family B GPCRs and thereby affect fundamental characteristics of these receptors. These RAMP-mediated effects include alteration of cell-surface expression and changes to the receptor's ligand binding profile and intracellular signaling [4]. The best-characterized of these RAMP-mediated effects are with the receptors CLR and CTR [7].…”

Section: Discussionmentioning

confidence: 99%

“…In mammals, RAMPs form a family of three single-pass membrane proteins (RAMP1, RAMP2, RAMP3). They have a range of effects on GPCRs including promotion of cell-surface expression plus modulation of ligand binding profile and signal transduction pathways [4]. The archetypical RAMP:GPCR interaction is that between a RAMP and the calcitonin receptor-like receptor (CLR) where there is direct interaction between the two proteins [5,6].…”

Section: Introductionmentioning

confidence: 99%

Interactions between RAMP2 and CRF receptors: The effect of receptor subtypes, splice variants and cell context

Bailey¹,

Harris²,

Barkan³

et al. 2019

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Corticotrophin releasing factor (CRF) acts via two family B G-protein-coupled receptors, CRFR1 and CRFR2. Additional subtypes exist due to alternative splicing. CRFR1is the most widely expressed subtype and lacks a 29-residue insert in the first intracellular loop that is present in CRFR1. It has been shown previously that co-expression of CRFR1 with receptor activity modifying protein 2 (RAMP2) in HEK 293S cells increased the cell-surface expression of both proteins suggesting a physical interaction as seen with RAMPs and calcitonin receptorlike receptor (CLR).This study investigated the ability of CRFR1CRFR1 and CRFR2 to promote cell-surface expression of FLAG-tagged RAMP2. Four different cell-lines were utilised to investigate the effect of varying cellular context; COS-7, HEK 293T, HEK 293S and [CTR]HEK 293 (which lacks endogenous calcitonin receptor). In all cell-lines, CRFR1and CRFR1 enhanced RAMP2 cell-surface expression. The magnitude of the effect on RAMP2was dependent on the cell-line ([CTR]HEK 293>COS-7>HEK 293T>HEK 293S). RT-PCR indicated this variation may relate to differences in endogenous RAMP expression between cell types. Furthermore, pre-treatment with CRF resulted in a loss of cell-surface FLAG-RAMP2 when it was co-expressed with CRFR1 subtypes. CRFR2 co-expression had no effect on RAMP2 in any cell-line. Molecular modelling suggests that the potential contact interface between the extracellular domains of RAMP2 and CRF receptor subtypes is smaller than that of RAMP2 and CRL, the canonical receptor:RAMP pairing, assuming a physical interaction.Furthermore, a specific residue difference between CRFR1 subtypes (glutamate) and CRFR2 (histidine) in this interface region may impair CRFR2RAMP2 interaction by electrostatic repulsion.

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The effects of RAMPs upon cell signalling

Cited by 15 publications

References 64 publications

Multiplexed analysis of the secretin-like GPCR-RAMP interactome

Multiplexed analysis of the secretin-like GPCR-RAMP interactome

Multiplexed Analysis of the Secretin-like GPCR-RAMP Interactome

Interactions between RAMP2 and CRF receptors: The effect of receptor subtypes, splice variants and cell context

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