TABLE 2.

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)