Summary of experimental methods used to identify GPCR–RAMP interactions Studies are sorted chronologically.
| Study | Method | Looked for | GPCR–RAMP tested | Also looked at |
|---|---|---|---|---|
| McLatchie et al. (1998) | Expression cloning | Signaling in response to CGRP | Discovered CALCRL–RAMP1,2,3 | Ligand binding, FACS for RAMP and CALCRL surface expression, immunoblot (crosslinking, radioligand labeling, glycosylase treatment) |
| Christopoulos et al. (1999) | Radioligand binding | Increased binding of amylin and salmon CT upon RAMP expression | Discovered CALCR–RAMP1,2,3 | Competition assay for peptide radioligand binding, cAMP response, immunoblot (crosslinking), IF for RAMP1 localization |
| Christopoulos et al. (2003) | IF | Increased RAMP surface expression | Interacting: VIPR1–RAMP1,2,3 GCGR–RAMP2 PTH1R–RAMP2 PTH2R–RAMP3 Noninteracting: GHRH, VIPR2, GLP1R, GLP2R | VIPR1 signaling (cAMP, PI), radioligand binding |
| Bouschet et al. (2005) | IF | Increased GPCR surface expression GPCR–RAMP colocalization | Interacting: CaSR–RAMP1,3 Noninteracting: CaSR–RAMP2 | Surface biotinylation, siRNA KD of RAMP1 (look at CaSR surface expression), co-IP, IF of GPCR with different cell compartment markers, GPCR glycosylation changes |
| Harikumar et al. (2009) | Fluorescence, BRET | Increased RAMP surface expression Saturating BRET signal that increases then plateaus. | Interacting: SCTR–RAMP3 Noninteracting: SCTR–RAMP1,2 | Bimolecular fluorescence complementation (look for fluorescence at the PM), assays with truncation and chimeric mutants of SCTR with WT or truncation mutants of RAMP, SCTR signaling (cAMP, Ca2+ flux, ERK1/2 phosphorylation) |
| Wootten et al. (2013) | ELISA | Increased RAMP surface expression Increased GPCR surface expression | Interacting: VIPR2–RAMP1,2,3, CRHR1–RAMP2 Noninteracting: GLP1R | G-protein binding to GPCR, GPCR signaling (cAMP, Ca2+ flux), in vivo experiment (measured plasma levels of adrenocorticotropic hormone in RAMP2+/− mice) |
| Lenhart et al. (2013) | IF BRET | Increased RAMP surface expression and colocalization with GPCR, Saturating BRET signal that increases then plateaus | Interacting: GPR30–RAMP3 | Co-IP, expression changes in vivo, localization, changes in vivo, in vivo experiment (studied cardiac fibrosis and left ventricular hypertrophy in RenTgMk; RAMP3+/+ and RAMP3−/− mice) |
| Cegla et al. (2017) (Note: GPCR–RAMP interaction was previously published) | IF | Changes in GPCR surface expression GPCR–RAMP co-localization | GCGR–RAMP2 | Radioligand binding, GPCR signaling with RAMP2 overexpression or siRNA KD (cAMP, Ca2+ flux, β-arrestin1 recruitment) |
| Barbash et al. (2019) | MERFISH | Significant changes in GPCR expression at the mRNA level upon RAMP2 co-expression | 14 GPCRs: ADORA2B, S1PR1, NTSR1, OPRD1, F2RL3, GPR50, GPR141, GPR160, GPR176, LGR4, P2YR8, CELSR2, FZD1 (tested with RAMP2 only) | Bioinformatics comparison with phylogenic correlation coefficient |
| Bailey et al. (2019) | FACS ELISA | Increased RAMP surface expression (FACS) Increased GPCR surface expression (ELISA) | Interacting: CRHR1α, CRHR1β–RAMP2 Noninteracting: CRHR2β–RAMP2 | GPCR signaling (cAMP), GPCR and RAMP expression (mRNA), molecular modeling of interaction interface |
| Lorenzen et al. (2019) | SBA | High median fluorescence intensity signal relative to control | Overview: 15 Secretin GPCRs plus ACKR3, ADGRF5, CCR5, CCR7, CXCR3, CXCR4, GPR4, GPR182 (with all 3 RAMPs) | PLA |
| Mackie et al. (2019) | BRET FACS | Saturating BRET signal that increases then plateaus Increased RAMP surface expression | Overview: 24 Chemokine GPCRs (with all 3 RAMPs) | PLA, co-IF with different biomarkers, ACKR3 signaling (cAMP), coculture scavenging activity assay, in vivo experiment (retinal angiogenesis in Admhi/hi, Adm+/+, Ackr3+/−, Ackr3−/−, RAMP3+/+, and RAMP3−/− mice) |
| Shao et al. (2022) | BRET IF | Saturating BRET signal that increases then plateaus Altered RAMP surface expression and colocalization, | Overview: 7 glucagon family GPCRs (with all 3 RAMPs) | GPCR signaling (cAMP, Gαq activation, β-arrestin1 and β-arrestin2 recruitment) |