Elucidating the Interactome of G Protein-Coupled Receptors and Receptor Activity-Modifying Proteins

Abstract

G protein-coupled receptors (GPCRs) are known to interact with several other classes of integral membrane proteins that modulate their biology and pharmacology. However, the extent of these interactions and the mechanisms of their effects are not well understood. For example, one class of GPCR-interacting proteins, receptor activity-modifying proteins (RAMPs), comprise three related and ubiquitously expressed single-transmembrane span proteins. The RAMP family was discovered more than two decades ago, and since then GPCR–RAMP interactions and their functional consequences on receptor trafficking and ligand selectivity have been documented for several secretin (class B) GPCRs, most notably the calcitonin receptor-like receptor. Recent bioinformatics and multiplexed experimental studies suggest that GPCR–RAMP interactions might be much more widespread than previously anticipated. Recently, cryo-electron microscopy has provided high-resolution structures of GPCR–RAMP–ligand complexes, and drugs have been developed that target GPCR–RAMP complexes. In this review, we provide a summary of recent advances in techniques that allow the discovery of GPCR–RAMP interactions and their functional consequences and highlight prospects for future advances. We also provide an up-to-date list of reported GPCR–RAMP interactions based on a review of the current literature. Significance Statement Receptor activity-modifying proteins (RAMPs) have emerged as modulators of many aspects of G protein-coupled receptor (GPCR)biology and pharmacology. The application of new methodologies to study membrane protein–protein interactions suggests that RAMPs interact with many more GPCRs than had been previously known. These findings, especially when combined with structural studies of membrane protein complexes, have significant implications for advancing GPCR-targeted drug discovery and the understanding of GPCR pharmacology, biology, and regulation.

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

“…Interestingly, we observed a strong attenuation of MRGPRX4 surface expression when it was co-expressed with RAMP2, an effect that scaled with the amount of RAMP2 expressed. CALCRL expressed alone or with RAMP2 served as a negative and positive control, respectively, and as expected CALCRL surface expression strongly increased upon RAMP2 co-expression 14,15 (Fig. 6c).…”

“…We hypothesized that RAMPs may be a missing component in our understanding of MRGPRX4 and applied an SBA assay to test for MRGPRX4-RAMP interactions. MRGPRX4 is not the first class A GPCR that has been shown to interact with RAMPs 15 , however it is the first delta subfamily class A GPCR with RAMP interactions that have been both identified and functionally characterized. The gamma subfamily class A GPCR atypical chemokine receptor 3 (ACKR3) was recently shown to interact with the RAMPs, with distinct functional consequences for the ACKR3-RAMP3 interaction 23 .…”

“…RAMP2 has been reported to have pleiotropic effects on G protein activation and β-arrestin recruitment 15 . For example, it promotes β-arrestin recruitment to CALCRL and parathyroid hormone 1 receptor (PTH1R) but has no effect on β-arrestin recruitment to receptors such as the secretin receptor (SCTR) 15, 24–26 .…”

“…However, none of these have been shown to be ubiquitination sites. RAMP2 has one C-terminal lysine, but there are conflicting reports regarding whether it is a ubiquitination site or not 15 .…”

“…Therefore, the modulatory role of RAMP3, if any, on MRGPRX4 is still unknown. RAMP3 has a unique C-terminal tail motif compared with the other RAMPs and has been shown to affect the downstream trafficking of several interacting receptors, such as CALCRL and the atypical chemokine receptor 3, ACKR3 15,23,[32][33][34] . Therefore, the elucidation of the mode of MRGPRX4 desensitization, degradation and recycling in the presence or absence of each RAMP may reveal unique effects of RAMP3.…”