Summary of experimental methods used to identify GPCR–RAMP interactions Studies are sorted chronologically.

StudyMethodLooked forGPCR–RAMP testedAlso looked at
McLatchie et al. (1998)Expression cloningSignaling in response to CGRPDiscovered CALCRL–RAMP1,2,3Ligand binding, FACS for RAMP and CALCRL surface expression, immunoblot (crosslinking, radioligand labeling, glycosylase treatment)
Christopoulos et al. (1999)Radioligand bindingIncreased binding of amylin and salmon CT upon RAMP expressionDiscovered CALCR–RAMP1,2,3Competition assay for peptide radioligand binding, cAMP response, immunoblot (crosslinking), IF for RAMP1 localization
Christopoulos et al. (2003)IFIncreased RAMP surface expressionInteracting: VIPR1–RAMP1,2,3
Noninteracting: GHRH, VIPR2, GLP1R, GLP2R
VIPR1 signaling (cAMP, PI), radioligand binding
Bouschet et al. (2005)IFIncreased 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, BRETIncreased 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)ELISAIncreased 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
Increased RAMP surface expression and colocalization with GPCR,
Saturating BRET signal that increases then plateaus
Interacting: GPR30–RAMP3Co-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)
IFChanges in GPCR surface expression
GPCR–RAMP co-localization
GCGR–RAMP2Radioligand binding, GPCR signaling with RAMP2 overexpression or siRNA KD (cAMP, Ca2+ flux, β-arrestin1 recruitment)
Barbash et al. (2019)MERFISHSignificant changes in GPCR expression at the mRNA level upon RAMP2 co-expression14 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
Increased RAMP surface expression (FACS)
Increased GPCR surface expression (ELISA)
Interacting: CRHR1α, CRHR1β–RAMP2
GPCR signaling (cAMP), GPCR and RAMP expression (mRNA), molecular modeling of interaction interface
Lorenzen et al. (2019)SBAHigh median fluorescence intensity signal relative to controlOverview: 15 Secretin GPCRs plus ACKR3, ADGRF5, CCR5, CCR7, CXCR3, CXCR4, GPR4, GPR182 (with all 3 RAMPs)PLA
Mackie et al. (2019)BRET
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
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)