Loss of receptor activity-modifying protein 2 in mice causes placental dysfunction and alters PTH1R regulation

Kadmiel, Mahita; Matson, Brooke C.; Espenschied, Scott T.; Lenhart, Patricia M.; Caron, Kathleen M.

doi:10.1371/journal.pone.0181597

Cited by 12 publications

(11 citation statements)

References 32 publications

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“…Here, we investigated possible modulatory effects using the class B PTH1R as a model system. Coexpression of RAMP isoforms with PTH1R in HEK293 cells mimics the physiological context in which both receptor and RAMPs are abundantly expressed ( 30 ); these conditions make a confounding contribution of the low levels of endogenous RAMPs in HEK293 cells negligible. Among the three RAMPs, this receptor showed a clear preference for RAMP2.…”

Section: Discussionmentioning

confidence: 99%

“…It is controversial whether PTH1R interacts only or preferentially with RAMP2 ( 28 ) or all three RAMPs ( 28 , 29 ). In RAMP2 knock-out mice, PTH1R function is deregulated, and placental dysfunction is observed ( 30 ), suggesting a major physiological role of the PTH1R/RAMP2 interaction. Yet, the molecular mechanisms of how RAMPs may modulate the activation dynamics of PTH1R and their signaling properties remain to be elucidated.…”

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confidence: 99%

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Functional modulation of PTH1R activation and signaling by RAMP2

Nemec

Schihada

Kleinau

et al. 2022

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

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Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein–coupled receptors (GPCRs), including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function. Using different optical biosensors to measure the activation of PTH1R and its downstream signaling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique preactivated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signaling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signaling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered β-arrestin2 recruitment to PTH1R. Employing homology modeling, we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signaling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Functional modulation of PTH1R activation and signaling by RAMP2

Nemec

Schihada

Kleinau

et al. 2022

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

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“…A single GPCR from the glutamate-like family, calcium-sensing receptor (CaSR), and a single GPCR from the rhodopsinlike family, G protein-coupled estrogen receptor 1 (GPER1), have been reported to interact with at least one RAMP (4)(5)(6). Several lines of evidence from disease-associated pathologies, tissue expression, and knockout mice indicate that the breadth of GPCR-RAMP interactions is not yet fully appreciated (13,14). Furthermore, we demonstrated recently that GPCRs and RAMPs globally coevolved and display correlated mRNA levels across human tissues, which suggests the likelihood for more widespread GPCR-RAMP interactions (15).…”

Section: Introductionmentioning

confidence: 99%

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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“…A single GPCR from the glutamate-like family, calcium-sensing receptor (CaSR), and a single GPCR from the rhodopsin-like family, G protein-coupled estrogen receptor 1 (GPER1), have been reported to interact with at least one RAMP (4–6) . 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) .…”

Section: Introductionmentioning

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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Loss of receptor activity-modifying protein 2 in mice causes placental dysfunction and alters PTH1R regulation

Cited by 12 publications

References 32 publications

Functional modulation of PTH1R activation and signaling by RAMP2

Functional modulation of PTH1R activation and signaling by RAMP2

Multiplexed analysis of the secretin-like GPCR-RAMP interactome

Multiplexed Analysis of the Secretin-like GPCR-RAMP Interactome

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