Itch receptor MRGPRX4 interacts with the receptor activity-modifying proteins (RAMPs)

Abstract

Cholestatic itch is a severe and debilitating symptom in liver diseases with limited treatment options. The class A G protein-coupled receptor (GPCR) Mas-related GPCR subtype X4 (MRGPRX4) has been identified as a receptor for bile acids, which are potential cholestatic pruritogens. An increasing number of GPCRs have been shown to interact with receptor activity-modifying proteins (RAMPs), which can modulate different aspects of GPCR biology. Using a combination of multiplexed immunoassay and proximity ligation assay we show that MRGPRX4 interacts with RAMPs. The interaction of MRGPRX4 with RAMP2, but not RAMP1 or 3, causes attenuation of basal and agonist-dependent signaling, which correlates with a decrease of MRGPRX4 cell surface expression as measured using a quantitative NanoBRET pulse-chase assay. Finally, we use AlphaFold Multimer to predict the structure of the MRGPRX4-RAMP2 complex. The discovery that RAMP2 regulates MRGPRX4 may have direct implications for future drug development for cholestatic itch.

MRGPRX4 mediates phospho-drug–associated pruritus in a humanized mouse model

“…Nonetheless, the observed behavioral phenotype may be exaggerated or skewed by the nonphysiologic amounts of receptor expression. A recent study identified the presence of human receptor activity–modifying proteins (RAMPs) that modulate MRGPRX4 expression ( 43 ). It is unclear whether MRGPRX4 can also be regulated by the murine RAMP counterpart.…”

“…Given our demonstrated structural differences between S83 and the previously published L83, even individual amino acid changes in this highly variable sequence can have profound impacts on receptor activation. Furthermore, RAMP2 was reported to down-regulate the surface expression of MRGPRX4 ( 43 ). This finding highlights a regulatory mechanism that could also shape the representation of itch.…”

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

“…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).…”