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

Ilana B. Kotliar; Emily Lorenzen; Jochen M. Schwenk; Debbie L. Hay; Thomas P. Sakmar

doi:10.1124/pharmrev.120.000180

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Fig. 1
GPCR classifications based on phylogenetic analysis. The phylogenetic tree positions of GPCRs tested for RAMP interactions are indicated. Adapted from Lv et al. (2016).
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Fig. 2
Summary of how RAMPs modulate GPCR biology. The four major regulatory effects of RAMPs on RAMP-interacting GPCRs are shown, with techniques commonly used to interrogate the regulatory effect or the presence of the complex in green text bubbles.
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Fig. 3
Schematic highlighting the most common methodologies used to identify GPCR–RAMP interactions.
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Fig. 4
Structure of CALCRL in complex with RAMP1 and CGRP obtained from cryo-EM (PDB code: 6E3Y) (Liang et al., 2018). Center panel: CALCRL, rainbow color gradation from deep blue (N-terminal tail) to deep red (C-terminal tail); RAMP1, magenta; CGRP, gray. The original published structure also included the bound G protein, which has been removed here for clarity. The N-terminal tails and the ECDs of CALCRL and RAMP1 are at the top, while their C-terminal tails and intracellular domains (ICDs) are at the bottom. The C-terminal residues of RAMP1 that stretch beyond the membrane bilayer are not resolved (dashed line). All residues of RAMPs that interact with CALCRL as determined by all recent cryo-EM structures are shown as sticks in the center and right-side insets (Liang et al., 2018; Liang et al., 2020a; Josephs et al., 2021). Similarly, residues of CALCRL that may interact with either RAMP1, 2, or 3 are shown as sticks in the center panel and right-side insets. In the right-side insets, any atoms between CALCRL and RAMP1 that are within 4 Å of each other are marked with yellow dashed lines. The left-side boxed structure shows interacting residues as spheres to highlight the potential CALCRL–RAMP interaction interface. All RAMP amino acid residues with potential interactions with CALCRL are shown as magenta spheres. Amino acid residues in the TM, ECL, and ECD regions of CALCRL that potentially interact with RAMPs are shown as blue spheres.

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TABLE 1

Reports of GPCRs tested for RAMP interaction, followed by summary statistics GPCRs are grouped by family and sorted alphabetically within each family. Key references are provided.

GPCR	Abbreviation	Uniprot	Family	RAMP
Adhesion G-protein coupled receptor F5	ADGRF5	Q8IZF4	Adhesion	RAMP3 (Lorenzen et al., 2019)
Cadherin EGF LAG seven-pass G-type receptor 2	CELSR2	Q9HCU4	Adhesion	None (only RAMP2 tested) (Barbash et al., 2019)
Frizzled family receptor 1	FZD1	Q9UP38	Frizzled	None (only RAMP2 tested) (Barbash et al., 2019)
Calcium-sensing receptor	CaSR	P41180	Glutamate	RAMP1 and 3 (Bouschet et al., 2008; Bouschet et al., 2005; Desai et al., 2014)
Pituitary adenylate-cyclase activating polypeptide type 1	ADYCAP1R1	P41586	Secretin	RAMP1, 2, and 3 (Lorenzen et al., 2019) RAMP2 and 3 (M. Harris et al., preprint, DOI: https://doi.org/10.1101/2021.04.08.436756)
Calcitonin receptor-like receptor	CALCRL	Q16602	Secretin	RAMP1, 2 and 3 (McLatchie et al., 1998) and many others (including solved structures)
Calcitonin receptor	CALCR	P30988	Secretin	RAMP1, 2 and 3 (Armour et al., 1999; Christopoulos et al., 1999) and many others
Corticotropin-releasing hormone receptor 1	CRHR1	P34998	Secretin	None (Tasma et al., 2020) RAMP2 (Wootten et al., 2013; Wootten et al., 2013;Bailey et al., 2019) RAMP3 (Lorenzen et al., 2019) RAMP2,3 (M. Harris et al., preprint)
Corticotropin-releasing hormone receptor 2	CRHR2	Q13324	Secretin	None (Lorenzen et al., 2019; Tasma et al., 2020) None (only RAMP2 tested) (Bailey et al., 2019) RAMP2,3 (M. Harris et al., preprint)
Glucagon receptor	GCGR	P47871	Secretin	RAMP2 (Christopoulos et al., 2003; Weston et al., 2015; Cegla et al., 2017; McGlone et al., 2021) RAMP1,3 (Lorenzen et al., 2019) RAMP1,2,3 (Shao et al., 2022; M. Harris et al., preprint)
Growth hormone-releasing hormone	GHRHR	Q02643	Secretin	None (Christopoulos et al., 2003) RAMP2,3 (Lorenzen et al., 2019) RAMP1,2,3 (Shao et al., 2022; M. Harris et al., preprint) RAMP1,2 with splice variant 1 of GHRHR (Shao et al., 2022)
Gastric inhibitory polypeptide receptor	GIPR	P48546	Secretin	RAMP1,2,3 (Lorenzen et al., 2019; Shao et al., 2022; M. Harris et al., preprint)
Glucagon-like peptide 1 receptor	GLP1R	P43220	Secretin	None (Christopoulos et al., 2003; Wootten et al., 2013) RAMP2,3 (Shao et al., 2022) RAMP1,2,3 (Lorenzen et al., 2019) (M. Harris et al., preprint)
Glucagon-like peptide 2 receptor	GLP2R	O95838	Secretin	None (Christopoulos et al., 2003) RAMP1,2,3 (Lorenzen et al., 2019; M. Harris et al., preprint) RAMP3 (Shao et al., 2022)
Parathyroid hormone 1 receptor	PTH1R	Q03431	Secretin	RAMP2 (Christopoulos et al., 2003; Nemec et al., 2022) RAMP1,3 (Lorenzen et al., 2019) RAMP2,3 (M. Harris et al., preprint) RAMP3 (Phelps et al., 2005)
Parathyroid hormone 2 receptor	PTH2R	P49190	Secretin	RAMP3 (Christopoulos et al., 2003) RAMP1,2 (Lorenzen et al., 2019) RAMP1,2,3 (M. Harris et al., preprint)
Secretin receptor	SCTR	P47872	Secretin	RAMP3 (Harikumar et al., 2009) RAMP1,2 (Lorenzen et al., 2019) RAMP1,2,3 (Shao et al., 2022; M. Harris et al., preprint)
VIP and PACAP receptor 1	VIPR1	P32241	Secretin	RAMP1,2,3 (Christopoulos et al., 2003) (M. Harris et al., preprint) RAMP2,3 (Lorenzen et al., 2019)
VIP and PACAP receptor 2	VIPR2	P41587	Secretin	RAMP1,2,3 (Wootten et al., 2013; (M. Harris et al., preprint) RAMP2,3 (Lorenzen et al., 2019)
Atypical chemokine receptor 1	ACKR1	Q16570	Rhodopsin	RAMP1,2,3 (Mackie et al., 2019)
Atypical chemokine receptor 2	ACKR2	O00590	Rhodopsin	RAMP1,3 (Mackie et al., 2019)
Atypical chemokine receptor 3	ACKR3	P25106	Rhodopsin	RAMP2,3 (Lorenzen et al., 2019) RAMP1,2,3 (Mackie et al., 2019)
Atypical chemokine receptor 4	ACKR4	Q9NPB9	Rhodopsin	RAMP2,3 (Mackie et al., 2019)
Atypical chemokine receptor 5	ACKR5	O00421	Rhodopsin	None (Mackie et al., 2019)
Adenosine A2B receptor	ADORA2B	P29275	Rhodopsin	RAMP2 (Barbash et al., 2019)
Beta 2 adrenergic receptor	B2ADR	P07550	Rhodopsin	None (Mackie et al., 2019; Shao et al., 2022; M. Harris et al., preprint)
C-C Chemokine receptor type 1	CCR1	P32246	Rhodopsin	RAMP1,2,3 (Mackie et al., 2019)
C-C Chemokine receptor type 2	CCR2	P41597	Rhodopsin	RAMP2,3 (Mackie et al., 2019)
C-C Chemokine receptor type 3	CCR3	P51677	Rhodopsin	RAMP1,2,3 (Mackie et al., 2019)
C-C Chemokine receptor type 4	CCR4	P51679	Rhodopsin	RAMP1,2,3 (Mackie et al., 2019)
C-C Chemokine receptor type 5	CCR5	P51681	Rhodopsin	None (Lorenzen et al., 2019) RAMP2,3 (Mackie et al., 2019)
C-C Chemokine receptor type 6	CCR6	P51684	Rhodopsin	RAMP1,2,3 (Mackie et al., 2019)
C-C Chemokine receptor type 7	CCR7	P32248	Rhodopsin	None (Lorenzen et al., 2019) RAMP3 (Mackie et al., 2019)
C-C Chemokine receptor type 8	CCR8	P51685	Rhodopsin	RAMP3 (Mackie et al., 2019)
C-C Chemokine receptor type 9	CCR9	P51686	Rhodopsin	RAMP1,3 (Mackie et al., 2019)
C-C Chemokine receptor type 10	CCR10	P46092	Rhodopsin	RAMP1,2,3 (Mackie et al., 2019)
Chemokine-like receptor 1	CMKLR1	Q99788	Rhodopsin	RAMP2,3 (Mackie et al., 2019)
Chemokine C-X3-C receptor 1	CX3CR1	P49238	Rhodopsin	RAMP1,2,3 (Mackie et al., 2019)
C-X-C chemokine receptor type 1	CXCR1	P25024	Rhodopsin	RAMP1,2 (Mackie et al., 2019)
C-X-C chemokine receptor type 2	CXCR2	P25025	Rhodopsin	RAMP2,3 (Mackie et al., 2019)
C-X-C chemokine receptor type 3	CXCR3	P49682	Rhodopsin	None (Lorenzen et al., 2019) RAMP3 (Mackie et al., 2019)
C-X-C chemokine receptor type 4	CXCR4	P61073	Rhodopsin	None (Lorenzen et al., 2019) RAMP1,3 (Mackie et al., 2019)
C-X-C chemokine receptor type 5	CXCR5	P32302	Rhodopsin	None (Mackie et al., 2019)
C-X-C chemokine receptor type 6	CXCR6	O00574	Rhodopsin	RAMP3 (Mackie et al., 2019)
Proteinase-activated receptor 2	F2RL1	P55085	Rhodopsin	None (only RAMP2 tested) (Barbash et al., 2019)
Proteinase-activated receptor 2	F2RL3	Q96RI0	Rhodopsin	RAMP2 (Barbash et al., 2019)
G-protein coupled receptor 4	GRP4	P46093	Rhodopsin	RAMP1,2,3 (Lorenzen et al., 2019)
G protein-coupled estrogen receptor 1	GPR30	Q99527	Rhodopsin	RAMP3 (Lenhart et al., 2013)
Melatonin-related receptor	GPR50	Q15385	Rhodopsin	None (only RAMP2 tested) (Barbash et al., 2019)
Probably G protein-coupled receptor 141	GPR141	Q7Z602	Rhodopsin	RAMP2 (Barbash et al., 2019)
Probably G protein-coupled receptor 141	GPR160	Q9UJ42	Rhodopsin	None (only RAMP2 tested) (Barbash et al., 2019)
G protein-coupled receptor 176	GPR176	Q80WT4	Rhodopsin	None (only RAMP2 tested) (Barbash et al., 2019)
G-protein coupled receptor 182	GRP182	O15218	Rhodopsin	RAMP1,2,3 (Lorenzen et al., 2019)
Leucine-rich repeat-containing G-protein coupled receptor 4	LGR4	Q9BXB1	Rhodopsin	None (only RAMP2 tested) (Barbash et al., 2019)
P2Y purinoceptor 8	P2RY8	Q86VZ1	Rhodopsin	RAMP2 (Barbash et al., 2019)
Neurotensin receptor type 1	NTSR1	P30989	Rhodopsin	None (only RAMP2 tested) (Barbash et al., 2019)
δ-type opioid receptor	OPRD1	P41143	Rhodopsin	RAMP2 (Barbash et al., 2019)
Sphingosine 1-phosphate receptor 1	S1PR1	P21453	Rhodopsin	None (only RAMP2 tested) (Barbash et al., 2019)
Chemokine XC receptor 1	XCR1	P46094	Rhodopsin	None (Mackie et al., 2019)

Summary statistics	RAMP1	RAMP2	RAMP3
No. of GPCR interactors (at least one study)	28	30	41
	Secretin	Rhodopsin	Glutamate	Adhesion	Frizzled
No. of GPCRs that interact with any RAMP(s) (based on at least one report)	15	29	1	1	—
No. of GPCRs that don’t interact with any RAMP (based on at least one report)	5	8^a	—	1	1
No. of GPCRs in the family	15	719	22	33	11

Key references provided.
^aAnd 7 additional instances where the GPCR does not appear to interact with a RAMP, but only RAMP2 was tested.

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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 Adm^hi/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, Gα_q activation, β-arrestin1 and β-arrestin2 recruitment)

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TABLE 3

Published structures with RAMPs Asterisk (*) indicates structure obtained by cryo-electron microscopy. Otherwise, structures obtained by X-ray crystallography. All structures marked Full include the ECD and TM domains of the RAMP, with no or very poor C-terminal density observed. Stabilizing mutations made to the RAMP and/or GPCR are not listed but may be present.

GPCR	RAMP	Full length?	Relevant molecules present	Other molecules present	PDB Ref	Ref
None	RAMP1	ECD only		MSE	2YX8	Kusano et al. (2008)
None	RAMP2	ECD only	Ca⁺²		2XVT	Not published (Quigley, 2010)
None	RAMP2	ECD only		MSE	3AQE	Kusano et al. (2012)
CALCRL	RAMP1	ECD (both RAMP and GPCR)	Olcegepant	3N6, 3N7, sulfate ion	3N7S	ter Haar et al. (2010)
CALCRL	RAMP1	MBP–RAMP1 ECD–CALCRL ECD fusion	AM variant	Maltose	5V6Y	Booe et al. (2018)
CALCRL	RAMP1	MBP–RAMP1 ECD–CALCRL ECD fusion	AM2	Maltose, sodium ion	6D1U	Roehrkasse et al. (2018)
CALCRL	RAMP1	ECD + TM (no C terminus), CALCRL full length	CGRP, G_s heterotrimer	Nb35	6E3Y*	Liang et al. (2018a)
CALCRL	RAMP1	ECD (both RAMP and GPCR)	Telcagepant	3N6, N7R	3N7R	ter Haar et al. (2010)
CALCRL	RAMP1	ECD (both RAMP and GPCR)	(unliganded)	sulfate ion, MSE	3N7P	ter Haar et al. (2010)
CALCRL	RAMP1	MBP–RAMP1 ECD–CALCRL ECD fusion	CGRP analog	Maltose, magnesium ion	4RWG	Booe et al. (2015)
CALCRL	RAMP1	ECD (both RAMP and GPCR)	Erenumab (Fab)		6UMG	Garces et al. (2020)
CALCRL	RAMP1	MBP–RAMP1 ECD–CALCRL ECD fusion	Olcegepant	SN6, tetraethyl glycol (PG4), maltose, alpha-D-glucopyranose	6ZIS	Bucknell et al. (2020)
CALCRL	RAMP1	MBP–RAMP1 ECD–CALCRL ECD fusion	HTL22562	tetraethyl glycol (PG4), maltose, alpha-D-glucopyranose	6ZHO	Bucknell et al. (2020)
CALCRL	RAMP1	MBP–RAMP1 ECD–CALCRL ECD fusion	HTL0028125 (macrocycle)	Unknown (PDB entry on hold)	7P0F	Cansfield et al. (2022)
CALCRL	RAMP1	MBP–RAMP1 ECD–CALCRL ECD fusion	Macrocycle compound 13	Unknown (PDB entry on hold)	7P0I	Cansfield et al. (2022)
CALCRL	RAMP1	ECD + TM (no C terminus), CALCRL full length		Detergent micelle	7KNT*	Josephs et al. (2021)
CALCRL	RAMP1	ECD + TM (no C terminus), CALCRL full length	CGRP	Detergent micelle	7KNU*	Josephs et al. (2021)
CALCRL	RAMP2	ECD only		MSE	3AQF	Kusano et al. (2012)
CALCRL	RAMP2	MPB-RAMP2 ECD–CALCRL ECD fusion	AM	Maltose, 1,2-ethanediol	4RWF	Booe et al. (2015)
CALCRL	RAMP2	MPB–RAMP2 ECD–CALCRL ECD fusion	high-affinity AM (37-52) S45R/K46L/S48G/Q50W	alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose, maltose, formic acid, amino group	6V2E	Booe et al. (2020)
CALCRL	RAMP2	Full	AM, G_s heterotrimer	Nb35	6UUN*	Liang et al. (2020a)
CALCRL	RAMP3	Full	AM2, G_s heterotrimer	Nb35	6UVA*	Liang et al. (2020a)
CALCRL	RAMP3	Full	AM, G_s heterotrimer	Nb35	6UUS*	Liang et al. (2020a)
CALCR	RAMP1	Full	Rat amylin, G_s heterotrimer	Nb35, P42, phosphatidylethanolamine, cholesterol hemisuccinate, palmitic acid, 2-acetamido-2-deoxy-beta-D-glucopyranose	7TYF*	Cao et al. (2022)
CALCR	RAMP1	Full	Salmon CT, G_s heterotrimer	Nb35, cholesterol hemisuccinate, palmitic acid, 2-acetamido-2-deoxy-beta-D-glucopyranose	7TYW*	Cao et al. (2022)
CALCR	RAMP2	Full	Rat amylin, G_s heterotrimer	Nb35, cholesterol hemisuccinate, palmitic acid, 2-acetamido-2-deoxy-beta-D-glucopyranose	7TYX*	Cao et al. (2022)
CALCR	RAMP2	Full	Salmon CT, G_s heterotrimer	Nb35, cholesterol hemisuccinate, palmitic acid, 2-acetamido-2-deoxy-beta-D-glucopyranose	7TYY*	Cao et al. (2022)
CALCR	RAMP2	Full	CT, G_s heterotrimer	Nb35	7TYH*	Cao et al. (2022)
CALCR	RAMP3	Full	Rat amylin, G_s heterotrimer	Nb35, P42, cholesterol hemisuccinate, palmitic acid, 2-acetamido-2-deoxy-beta-D-glucopyranose	7TZF*	Cao et al. (2022)