Itch receptor MRGPRX4 interacts with the receptor activity–modifying proteins

Kotliar, Ilana B.; Ceraudo, Emilie; Kemelmakher-Liben, Kevin; Oren, Deena A.; Lorenzen, Emily; Dodig‐Crnković, Tea; Horioka, Mizuho; Huber, Thomas; Schwenk, Jochen M.; Sakmar, Thomas P.

doi:10.1016/j.jbc.2023.104664

Cited by 5 publications

(3 citation statements)

References 56 publications

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“…Knowing that fluorescently labeled chemokines covering a wide range of the light spectrum can be used as acceptors in NanoBRET provided the opportunity to conduct a multiplex NanoBRET binding assay for two chemokine receptors simultaneously for the first time. So far, multiplex-based assays have been applied to immunoassay-based protein-protein interactions [41], high-throughput GPCR antibody discovery [42,43], and the multiplexing of functional assays for GPCRs [44][45][46][47]. However, to the best of our knowledge, multiplex-based assays have not been used in the context of monitoring ligand-receptor-binding assays.…”

Section: Discussionmentioning

confidence: 99%

Multiplex Detection of Fluorescent Chemokine Binding to CXC Chemokine Receptors by NanoBRET

Adamska,

Leftheriotis,

Bosma

et al. 2024

IJMS

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NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously.

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Section: Discussionmentioning

confidence: 99%

Multiplex Detection of Fluorescent Chemokine Binding to CXC Chemokine Receptors by NanoBRET

Adamska,

Leftheriotis,

Bosma

et al. 2024

IJMS

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“…The plasmids encode an N-terminal 3xHA tag (amino acid sequence YPYDVPDYA) following the signal peptide (SP; amino acids 1 to 26, 1 to 42, and 1 to 27 for RAMP1, RAMP2, and RAMP3, respectively) and two C-terminal OLLAS tags (amino acid sequence SGFANELGPRLMGK) linked by a flexible spacer that also includes two Strep-tag II peptides (amino acid sequence WSHPQFEKGGGSGGGSGGGSWSHPQFEK). Each 3xHA-RAMP-OLLAS construct was generated using the TagMaster site-directed mutagenesis kit according to the manufacturer’s instructions for “long-range mutation” as previously reported ( 36 ) using the previously validated FLAG-RAMP-OLLAS construct as a starting point ( 9 ). All constructs were confirmed by sequencing in the forward and reverse directions (T7, BGHR primers) (Genewiz).…”

Section: Methodsmentioning

confidence: 99%

Multiplexed mapping of the interactome of GPCRs with receptor activity–modifying proteins

Kotliar,

Bendes,

Dahl

et al. 2024

Sci. Adv.

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Receptor activity–modifying proteins (RAMPs) form complexes with G protein–coupled receptors (GPCRs) and may regulate their cellular trafficking and pharmacology. RAMP interactions have been identified for about 50 GPCRs, but only a few GPCR-RAMP complexes have been studied in detail. To elucidate a comprehensive GPCR-RAMP interactome, we created a library of 215 dual epitope-tagged (DuET) GPCRs representing all GPCR subfamilies and coexpressed each GPCR with each of the three RAMPs. Screening the GPCR-RAMP pairs with customized multiplexed suspension bead array (SBA) immunoassays, we identified 122 GPCRs that showed strong evidence for interaction with at least one RAMP. We screened for interactions in three cell lines and found 23 endogenously expressed GPCRs that formed complexes with RAMPs. Mapping the GPCR-RAMP interactome expands the current system-wide functional characterization of RAMP-interacting GPCRs to inform the design of selective therapeutics targeting GPCR-RAMP complexes.

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“… 107 Further, MRGPRX4 has been shown to associate with receptor activating-modifying protein-2 (RAMP-2), but not other members of the RAMP family. 113 RAMP-2 reduces the surface expression of MRGPRX4 ( Fig. 3 ), as well as inhibits basal and agonist-induced signaling driven by the MRGPRX4 ligands nateglinide 114 and bile acids.…”

Section: Introductionmentioning

confidence: 99%