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Adrenomedullin 2/intermedin is a slow off-rate, long-acting endogenous agonist of the adrenomedullin2 G protein–coupled receptor

2023; Elsevier BV; Volume: 299; Issue: 6 Linguagem: Inglês

10.1016/j.jbc.2023.104785

ISSN

1083-351X

Autores

Katie M. Babin, Jordan A. Karim, Peyton H. Gordon, James Lennon, Alex Dickson, Augen A. Pioszak,

Tópico(s)

Cancer, Stress, Anesthesia, and Immune Response

Resumo

Adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and calcitonin gene-related peptide (CGRP) have functions in the cardiovascular, lymphatic, and nervous systems by activating three heterodimeric receptors comprising the class B GPCR CLR and a RAMP1, -2, or -3 modulatory subunit. CGRP and AM prefer the RAMP1 and RAMP2/3 complexes, respectively, whereas AM2/IMD is thought to be relatively nonselective. Accordingly, AM2/IMD exhibits overlapping actions with CGRP and AM, so the rationale for this third agonist for the CLR–RAMP complexes is unclear. Here, we report that AM2/IMD is kinetically selective for CLR–RAMP3, known as the AM2R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD–AM2R elicited longer-duration cAMP signaling than the other peptide–receptor combinations. AM2/IMD and AM bound the AM2R with similar equilibrium affinities, but AM2/IMD had a slower off-rate and longer receptor residence time, thus explaining its prolonged signaling capacity. Peptide and receptor chimeras and mutagenesis were used to map the regions responsible for the distinct binding and signaling kinetics to the AM2/IMD mid-region and the RAMP3 extracellular domain (ECD). Molecular dynamics simulations revealed how the former forms stable interactions at the CLR ECD–transmembrane domain interface and how the latter augments the CLR ECD binding pocket to anchor the AM2/IMD C terminus. These strong binding components only combine in the AM2R. Our findings uncover AM2/IMD–AM2R as a cognate pair with unique temporal features, reveal how AM2/IMD and RAMP3 collaborate to shape CLR signaling, and have significant implications for AM2/IMD biology. Adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and calcitonin gene-related peptide (CGRP) have functions in the cardiovascular, lymphatic, and nervous systems by activating three heterodimeric receptors comprising the class B GPCR CLR and a RAMP1, -2, or -3 modulatory subunit. CGRP and AM prefer the RAMP1 and RAMP2/3 complexes, respectively, whereas AM2/IMD is thought to be relatively nonselective. Accordingly, AM2/IMD exhibits overlapping actions with CGRP and AM, so the rationale for this third agonist for the CLR–RAMP complexes is unclear. Here, we report that AM2/IMD is kinetically selective for CLR–RAMP3, known as the AM2R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD–AM2R elicited longer-duration cAMP signaling than the other peptide–receptor combinations. AM2/IMD and AM bound the AM2R with similar equilibrium affinities, but AM2/IMD had a slower off-rate and longer receptor residence time, thus explaining its prolonged signaling capacity. Peptide and receptor chimeras and mutagenesis were used to map the regions responsible for the distinct binding and signaling kinetics to the AM2/IMD mid-region and the RAMP3 extracellular domain (ECD). Molecular dynamics simulations revealed how the former forms stable interactions at the CLR ECD–transmembrane domain interface and how the latter augments the CLR ECD binding pocket to anchor the AM2/IMD C terminus. These strong binding components only combine in the AM2R. Our findings uncover AM2/IMD–AM2R as a cognate pair with unique temporal features, reveal how AM2/IMD and RAMP3 collaborate to shape CLR signaling, and have significant implications for AM2/IMD biology. Adrenomedullin 2/intermedin (AM2/IMD) was discovered by two groups in 2004 based on its similarity to the calcitonin gene-related peptide (CGRP) family of peptides, which also includes adrenomedullin (AM) and amylin (1Roh J. Chang C.L. Bhalla A. Klein C. Hsu S.Y. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes.J. Biol. Chem. 2004; 279: 7264-7274Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 2Takei Y. Inoue K. Ogoshi M. Kawahara T. Bannai H. Miyano S. Identification of novel adrenomedullin in mammals: a potent cardiovascular and renal regulator.FEBS Lett. 2004; 556: 53-58Crossref PubMed Scopus (254) Google Scholar). AM2/IMD exhibits overlapping actions with CGRP and AM, but it is the least understood of these peptides. Like CGRP and AM, when administered peripherally AM2/IMD induces vasodilation, and it has protective effects in the cardiovascular, pulmonary, and renal systems (3Holmes D. Campbell M. Harbinson M. Bell D. Protective effects of intermedin on cardiovascular, pulmonary and renal diseases: comparison with adrenomedullin and CGRP.Curr. Protein Pept. Sci. 2013; 14: 294-329Crossref PubMed Scopus (36) Google Scholar). AM2/IMD stabilizes the endothelial barrier (4Aslam M. Pfeil U. Gunduz D. Rafiq A. Kummer W. Piper H.M. et al.Intermedin (adrenomedullin2) stabilizes the endothelial barrier and antagonizes thrombin-induced barrier failure in endothelial cell monolayers.Br. J. Pharmacol. 2012; 165: 208-222Crossref PubMed Scopus (50) Google Scholar), has anti-inflammatory actions (5Hagiwara M. Bledsoe G. Yang Z.R. Smith Jr., R.S. Chao L. Chao J. Intermedin ameliorates vascular and renal injury by inhibition of oxidative stress.Am. J. Physiol. Renal Physiol. 2008; 295: F1735-1743Crossref PubMed Scopus (48) Google Scholar), and protects against sepsis in mice (6Xiao F. Wang D. Kong L. Li M. Feng Z. Shuai B. et al.Intermedin protects against sepsis by concurrently re-establishing the endothelial barrier and alleviating inflammatory responses.Nat. Commun. 2018; 9: 2644Crossref PubMed Scopus (38) Google Scholar). AM2/IMD is angiogenic (7Smith Jr., R.S. Gao L. Bledsoe G. Chao L. Chao J. Intermedin is a new angiogenic growth factor.Am. J. Physiol. Heart Circ. Physiol. 2009; 297: H1040-1047Crossref PubMed Scopus (57) Google Scholar) and its knockout in mice revealed roles in enlarging the vascular lumen and promoting vessel fusion (8Kong L. Xiao F. Wang L. Li M. Wang D. Feng Z. et al.Intermedin promotes vessel fusion by inducing VE-cadherin accumulation at potential fusion sites and to achieve a dynamic balance between VE-cadherin-complex dissociation/reconstitution.MedComm (2020). 2020; 1: 84-102Crossref PubMed Scopus (5) Google Scholar, 9Wang L.J. Xiao F. Kong L.M. Wang D.N. Li H.Y. Wei Y.G. et al.Intermedin enlarges the vascular lumen by inducing the quiescent endothelial cell proliferation.Arterioscler. Thromb. Vasc. Biol. 2018; 38: 398-413Crossref PubMed Scopus (28) Google Scholar). Beyond the circulatory system, AM2/IMD protected against obesity and insulin resistance in mice by promoting beige cell biogenesis and reducing adipose inflammation (10Lv Y. Zhang S.Y. Liang X. Zhang H. Xu Z. Liu B. et al.Adrenomedullin 2 enhances beiging in white adipose tissue directly in an adipocyte-autonomous manner and indirectly through activation of M2 macrophages.J. Biol. Chem. 2016; 291: 23390-23402Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 11Pang Y. Li Y. Lv Y. Sun L. Zhang S. Li Y. et al.Intermedin restores hyperhomocysteinemia-induced macrophage polarization and improves insulin resistance in mice.J. Biol. Chem. 2016; 291: 12336-12345Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 12Zhang H. Zhang S.Y. Jiang C. Li Y. Xu G. Xu M.J. et al.Intermedin/adrenomedullin 2 polypeptide promotes adipose tissue browning and reduces high-fat diet-induced obesity and insulin resistance in mice.Int. J. Obes. (Lond). 2016; 40: 852-860Crossref PubMed Scopus (29) Google Scholar), and its central administration activated the hypothalamic–pituitary–adrenal axis and increased sympathetic activity (13Takahashi K. Morimoto R. Hirose T. Satoh F. Totsune K. Adrenomedullin 2/intermedin in the hypothalamo-pituitary-adrenal axis.J. Mol. Neurosci. 2011; 43: 182-192Crossref PubMed Scopus (37) Google Scholar, 14Taylor M.M. Bagley S.L. Samson W.K. Intermedin/adrenomedullin-2 acts within central nervous system to elevate blood pressure and inhibit food and water intake.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005; 288: R919-927Crossref PubMed Scopus (90) Google Scholar). There is promise in targeting AM2/IMD signaling for therapeutics for cardiovascular, metabolic, and other disorders (15Zhang S.Y. Xu M.J. Wang X. Adrenomedullin 2/intermedin: a putative drug candidate for treatment of cardiometabolic diseases.Br. J. Pharmacol. 2018; 175: 1230-1240Crossref PubMed Scopus (42) Google Scholar), but progress has been hindered by our limited understanding of the mechanisms that distinguish AM2/IMD signaling from that of AM and CGRP. AM2/IMD, AM, and CGRP share three heterodimeric receptors that comprise a common class B G protein–coupled receptor (GPCR) subunit, the calcitonin receptor-like receptor (CLR), and a variable receptor activity modifying protein (RAMP1-3) subunit that alters CLR ligand selectivity (16Pioszak A.A. Hay D.L. RAMPs as allosteric modulators of the calcitonin and calcitonin-like class B G protein-coupled receptors.Adv. Pharmacol. 2020; 88: 115-141Crossref PubMed Scopus (17) Google Scholar). These couple most efficiently to the Gs protein to activate cAMP signaling, but they also couple to Gq and Gi proteins (17Roehrkasse A.M. Warner M.L. Booe J.M. Pioszak A.A. Biochemical characterization of G protein coupling to calcitonin gene-related peptide and adrenomedullin receptors using a native PAGE assay.J. Biol. Chem. 2020; 295: 9736-9751Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 18Weston C. Winfield I. Harris M. Hodgson R. Shah A. Dowell S.J. et al.Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors.J. Biol. Chem. 2016; 291: 21925-21944Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Much of our understanding of the pharmacology of the three peptides and receptors comes from studies of cAMP signaling (19Hay D.L. Garelja M.L. Poyner D.R. Walker C.S. Update on the pharmacology of calcitonin/CGRP family of peptides: IUPHAR review 25.Br. J. Pharmacol. 2018; 175: 3-17Crossref PubMed Scopus (231) Google Scholar). CGRP is most potent at the CLR–RAMP1 complex, which is designated the CGRP receptor. This mediates CGRP actions in the trigeminovascular system and is the target of several recently approved inhibitor drugs for migraine headache (20Edvinsson L. Haanes K.A. Warfvinge K. Krause D.N. CGRP as the target of new migraine therapies - successful translation from bench to clinic.Nat. Rev. Neurol. 2018; 14: 338-350Crossref PubMed Scopus (525) Google Scholar). AM is most active and equally potent at the CLR–RAMP2 and CLR–RAMP3 complexes, which are termed the AM1 and AM2 receptors, respectively. The AM1 receptor mediates the essential developmental actions of AM in the cardiovascular and lymphatic systems (21Caron K.M. Smithies O. Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional adrenomedullin gene.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 615-619Crossref PubMed Scopus (319) Google Scholar, 22Dackor R. Fritz-Six K. Smithies O. Caron K. Receptor activity-modifying proteins 2 and 3 have distinct physiological functions from embryogenesis to old age.J. Biol. Chem. 2007; 282: 18094-18099Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 23Dackor R.T. Fritz-Six K. Dunworth W.P. Gibbons C.L. Smithies O. Caron K.M. Hydrops fetalis, cardiovascular defects, and embryonic lethality in mice lacking the calcitonin receptor-like receptor gene.Mol. Cell. Biol. 2006; 26: 2511-2518Crossref PubMed Scopus (115) Google Scholar, 24Ichikawa-Shindo Y. Sakurai T. Kamiyoshi A. Kawate H. Iinuma N. Yoshizawa T. et al.The GPCR modulator protein RAMP2 is essential for angiogenesis and vascular integrity.J. Clin. Invest. 2008; 118: 29-39Crossref PubMed Scopus (165) Google Scholar, 25Shindo T. Kurihara Y. Nishimatsu H. Moriyama N. Kakoki M. Wang Y. et al.Vascular abnormalities and elevated blood pressure in mice lacking adrenomedullin gene.Circulation. 2001; 104: 1964-1971Crossref PubMed Scopus (262) Google Scholar, 26Fritz-Six K.L. Dunworth W.P. Li M. Caron K.M. Adrenomedullin signaling is necessary for murine lymphatic vascular development.J. Clin. Invest. 2008; 118: 40-50Crossref PubMed Scopus (198) Google Scholar). Why cells need a second AM receptor is unclear as AM2R functions remain poorly defined. AM2/IMD is thought to be relatively nonselective for the three CLR–RAMP complexes (1Roh J. Chang C.L. Bhalla A. Klein C. Hsu S.Y. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes.J. Biol. Chem. 2004; 279: 7264-7274Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). It exhibits a slight preference for the AM2R, but the AM2 nomenclature is not meant to imply that it is the AM2/IMD receptor (19Hay D.L. Garelja M.L. Poyner D.R. Walker C.S. Update on the pharmacology of calcitonin/CGRP family of peptides: IUPHAR review 25.Br. J. Pharmacol. 2018; 175: 3-17Crossref PubMed Scopus (231) Google Scholar, 27Hong Y. Hay D.L. Quirion R. Poyner D.R. The pharmacology of adrenomedullin 2/intermedin.Br. J. Pharmacol. 2012; 166: 110-120Crossref PubMed Scopus (118) Google Scholar). In cAMP assays, AM2/IMD is slightly less potent than CGRP and AM at the CGRP and AM1 receptors, respectively, and is equipotent to AM at the AM2R. Different effects of AM2/IMD have been ascribed to signaling through either the CGRP or the AM receptors, but in many cases, the receptor(s) mediating a given AM2/IMD action is unclear. There are examples of apparent mismatch between AM2/IMD pharmacology at the cloned receptors and in vivo, but the bases for these discrepancies are unknown (27Hong Y. Hay D.L. Quirion R. Poyner D.R. The pharmacology of adrenomedullin 2/intermedin.Br. J. Pharmacol. 2012; 166: 110-120Crossref PubMed Scopus (118) Google Scholar). Recently, the idea that AM2/IMD, AM, and CGRP promote distinct receptor–transducer coupling profiles (agonist bias) has been explored. There are some data supporting this (18Weston C. Winfield I. Harris M. Hodgson R. Shah A. Dowell S.J. et al.Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors.J. Biol. Chem. 2016; 291: 21925-21944Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 28Clark A.J. Mullooly N. Safitri D. Harris M. de Vries T. MaassenVanDenBrink A. et al.CGRP, adrenomedullin and adrenomedullin 2 display endogenous GPCR agonist bias in primary human cardiovascular cells.Commun. Biol. 2021; 4: 776Crossref PubMed Scopus (13) Google Scholar), but other studies found less evidence for bias (17Roehrkasse A.M. Warner M.L. Booe J.M. Pioszak A.A. Biochemical characterization of G protein coupling to calcitonin gene-related peptide and adrenomedullin receptors using a native PAGE assay.J. Biol. Chem. 2020; 295: 9736-9751Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 29Garelja M.L. Au M. Brimble M.A. Gingell J.J. Hendrikse E.R. Lovell A. et al.Molecular mechanisms of class B GPCR activation: insights from adrenomedullin receptors.ACS Pharmacol. Transl. Sci. 2020; 3: 246-262Crossref PubMed Scopus (23) Google Scholar), so the extent to which signaling bias distinguishes the peptides remains unclear. Structural studies advanced our understanding of how the peptides bind the receptors and how the RAMPs modulate binding. In crystal structures of CLR–RAMP1/2–peptide ECD complexes, the C-terminal fragments of CGRP, AM, and AM2/IMD occupied a shared binding site on the CLR ECD, but with distinct, mostly unstructured conformations (30Booe J.M. Walker C.S. Barwell J. Kuteyi G. Simms J. Jamaluddin M.A. et al.Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor.Mol. Cell. 2015; 58: 1040-1052Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 31Roehrkasse A.M. Booe J.M. Lee S.M. Warner M.L. Pioszak A.A. Structure-function analyses reveal a triple beta-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design.J. Biol. Chem. 2018; 293: 15840-15854Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). RAMP1/2 augmented the ECD binding site with unique contacts to the peptides that contributed to ligand selectivity (30Booe J.M. Walker C.S. Barwell J. Kuteyi G. Simms J. Jamaluddin M.A. et al.Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor.Mol. Cell. 2015; 58: 1040-1052Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 32Booe J.M. Warner M.L. Pioszak A.A. Picomolar affinity antagonist and sustained signaling agonist peptide ligands for the adrenomedullin and calcitonin gene-related peptide receptors.ACS Pharmacol. Transl. Sci. 2020; 3: 759-772Crossref PubMed Scopus (6) Google Scholar, 33Booe J.M. Warner M.L. Roehrkasse A.M. Hay D.L. Pioszak A.A. Probing the mechanism of receptor activity-modifying protein modulation of GPCR ligand selectivity through rational design of potent adrenomedullin and calcitonin gene-related peptide antagonists.Mol. Pharmacol. 2018; 93: 355-367Crossref PubMed Scopus (34) Google Scholar). Cryo-EM structures of several peptide-bound CLR–RAMP complexes with Gs showed that the N-terminal half of each peptide adopted a disulfide loop and α-helix structure that similarly occupied the CLR TMD (34Liang Y.L. Belousoff M.J. Fletcher M.M. Zhang X. Khoshouei M. Deganutti G. et al.Structure and dynamics of adrenomedullin receptors AM1 and AM2 reveal key mechanisms in the control of receptor phenotype by receptor activity-modifying proteins.ACS Pharmacol. Transl. Sci. 2020; 3: 263-284Crossref PubMed Scopus (55) Google Scholar, 35Liang Y.L. Khoshouei M. Deganutti G. Glukhova A. Koole C. Peat T.S. et al.Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor.Nature. 2018; 561: 492-497Crossref PubMed Scopus (166) Google Scholar). 3D variability analyses of the cryo-EM data were consistent with differential RAMP1-3 modulation of CLR ECD–TMD interdomain dynamics playing a role in receptor phenotype (34Liang Y.L. Belousoff M.J. Fletcher M.M. Zhang X. Khoshouei M. Deganutti G. et al.Structure and dynamics of adrenomedullin receptors AM1 and AM2 reveal key mechanisms in the control of receptor phenotype by receptor activity-modifying proteins.ACS Pharmacol. Transl. Sci. 2020; 3: 263-284Crossref PubMed Scopus (55) Google Scholar). Despite this progress, several issues remain unresolved. How the two AM peptides bind the CLR–RAMP3 ECD complex is unclear because their C-terminal fragments in the AM2R cryo-EM structures were modeled with different conformations than in the crystal structures. The relative contributions of RAMP augmentation of the CLR ECD peptide binding site and modulation of CLR dynamics to receptor phenotype is unclear. In addition, it is unknown if the peptide agonists differentially modulate CLR dynamics and/or work in concert with the RAMPs to shape the signaling outcomes. The temporal features of AM2/IMD, AM, and CGRP signaling have received little attention. Here, we test the hypothesis that differences in their receptor binding and signaling kinetics distinguish their signaling capabilities. Strikingly, we find that AM2/IMD exhibits significantly longer-duration cAMP signaling at the AM2R than all other agonist-receptor pairings due to slow off-rate binding kinetics. We mapped the regions responsible for the slow off-rate to the AM2/IMD mid-region and to the RAMP3 ECD. Molecular dynamics (MD) simulations enabled by rebuilding the AM2R cryo-EM structures explained the structural basis for the distinct kinetics. Our results indicate that AM2/IMD–AM2R is a cognate pair and show how the peptide agonist and RAMP accessory protein collaborate to shape CLR signaling. We conclude that AM2/IMD is the endogenous agonist of the AM2R and suggest that its long-acting signaling at this receptor is the distinguishing feature of AM2/IMD. We measured cAMP in real time upon activation of each CLR–RAMP complex transiently expressed in COS-7 cells using the BRET cAMP biosensor CAMYEL (36Jiang L.I. Collins J. Davis R. Lin K.M. DeCamp D. Roach T. et al.Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway.J. Biol. Chem. 2007; 282: 10576-10584Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). COS-7 cells lack endogenous expression of CLR and RAMPs. The cells were stimulated at room temperature with 100 nM CGRP, AM, or AM2/IMD for 15 min followed by challenge with high-affinity CGRP or AM variant antagonist peptides (10 μM) that we recently developed (32Booe J.M. Warner M.L. Pioszak A.A. Picomolar affinity antagonist and sustained signaling agonist peptide ligands for the adrenomedullin and calcitonin gene-related peptide receptors.ACS Pharmacol. Transl. Sci. 2020; 3: 759-772Crossref PubMed Scopus (6) Google Scholar). The signal decay after antagonist addition provided a measure of signal duration, and we reasoned that it would act as a proxy for agonist dissociation. Each agonist gave a rise and fall to steady-state curve at each receptor in the absence of antagonist, and the BRET signal levels reflected the expected agonist potency rank orders (Fig. 1, A–C). Upon antagonist challenge, the cAMP BRET signal decayed quickly to baseline for each agonist at the CLR–RAMP1 and -2 complexes and for CGRP and AM at the CLR–RAMP3 complex (Fig. 1, A–C). In contrast, the cAMP signal for AM2/IMD at the CLR–RAMP3 complex decayed substantially slower (Fig. 1C). The decay phase of each curve was best fit by a one-phase exponential decay model (Fig. S1, A and B). The decay rates, half-lives, and time constants are summarized in Table S1, and the half-lives are shown in a scatter plot (Fig. 1D). The half-lives were within 0.6 to 3.5 min, except for AM2/IMD at AM2R, which exhibited a half-life of ∼17 min (Fig. 1D). A slower decay rate for AM2/IMD–AM2R was also observed at the physiological temperature of 37 °C (Fig. S1C). We extended these experiments to HEK293 cells. Our HEK293 cells exhibited a small endogenous response to CGRP in the absence of transfected receptors, but this was not large enough to confound the results with transfected receptors (Fig. S1D). No endogenous response was observed upon AM or AM2/IMD stimulation. The cAMP signaling kinetic profiles in HEK293 cells were similar to those in COS-7 cells (Fig. 1, E–H). Importantly, the slower decay of the AM2/IMD signal at AM2R was reproduced in the second cell line (t1/2 ∼ 13 min) (Table S1). These results indicated that the AM2/IMD–AM2R pairing is unique in exhibiting a long-duration cAMP signaling capability. To test if AM2/IMD binding kinetics were responsible for its long-acting signaling, we used nanoBRET technology (37Stoddart L.A. Kilpatrick L.E. Hill S.J. NanoBRET approaches to study ligand binding to GPCRs and RTKs.Trends Pharmacol. Sci. 2018; 39: 136-147Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) to compare the binding of AM and AM2/IMD to the AM2R. CLR was tagged with the BRET donor nanoluciferase (Nluc) at its N terminus, and we designed and ordered synthetic AM and AM2/IMD peptides labeled with the acceptor fluorophore TAMRA on a Lys residue substituted at equivalent positions in AM (N40) and AM2/IMD (D35) (Fig. 2A). These residues were chosen based on their solvent-exposed locations in the crystal and cryo-EM structures (30Booe J.M. Walker C.S. Barwell J. Kuteyi G. Simms J. Jamaluddin M.A. et al.Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor.Mol. Cell. 2015; 58: 1040-1052Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 31Roehrkasse A.M. Booe J.M. Lee S.M. Warner M.L. Pioszak A.A. Structure-function analyses reveal a triple beta-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design.J. Biol. Chem. 2018; 293: 15840-15854Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 34Liang Y.L. Belousoff M.J. Fletcher M.M. Zhang X. Khoshouei M. Deganutti G. et al.Structure and dynamics of adrenomedullin receptors AM1 and AM2 reveal key mechanisms in the control of receptor phenotype by receptor activity-modifying proteins.ACS Pharmacol. Transl. Sci. 2020; 3: 263-284Crossref PubMed Scopus (55) Google Scholar) and mutagenesis studies, which indicated that their substitution did not alter receptor ECD binding affinity (31Roehrkasse A.M. Booe J.M. Lee S.M. Warner M.L. Pioszak A.A. Structure-function analyses reveal a triple beta-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design.J. Biol. Chem. 2018; 293: 15840-15854Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 38Moad H.E. Pioszak A.A. Selective CGRP and adrenomedullin peptide binding by tethered RAMP-calcitonin receptor-like receptor extracellular domain fusion proteins.Protein Sci. 2013; 22: 1775-1785Crossref PubMed Scopus (26) Google Scholar). Wildtype pharmacology was observed for the Nluc–CLR–RAMP3 receptor with wildtype peptides (Fig. S2A) and for the TAMRA-labeled peptides at untagged CLR–RAMP3 (Fig. S2B) in cAMP accumulation assays. We further tested the labeled peptides in the real-time cAMP assay, which revealed wildtype behavior for AM–TAMRA (t1/2 ∼ 3 min) and a gain of function (slower decay) for AM2/IMD–TAMRA (t1/2 ∼ 43 min) (Fig. S2, C and D). These experiments indicated that the tagged receptor and peptides exhibited wildtype or near wildtype pharmacology and were thus suitable for nanoBRET studies. Nluc–CLR was coexpressed with RAMP3 in COS-7 cells and membranes were prepared for the binding studies, which were conducted at 25 °C and in the presence of GTPγS to uncouple the receptor from G protein. Equilibrium binding experiments revealed saturable binding of AM–TAMRA and AM2/IMD–TAMRA with binding affinities of 26 and 7 nM, respectively (Fig. 2B and Table S2). Varying the incubation time indicated that 3 h was sufficient to reach equilibrium (Fig. S2E). In association kinetics experiments AM–TAMRA reached equilibrium quicker than AM2/IMD–TAMRA (Fig. 2, C and D). Extended incubation revealed signal decay that we were unable to eliminate or correct for (Fig. S2, F and G), so we limited analysis of the association data to the first 10 min. The individual curves were best fit by a one-phase association exponential model and plots of the observed rates versus probe concentration showed a linear relationship consistent with a single-step binding model (Fig. 2E). The on- and off-rates were determined as the slope and y-intercept, respectively, as described (39Hoare S.R.J. Analyzing kinetic binding data.in: Markossian S. Grossman A. Brimacombe K. Arkin M. Auld D. Austin C. Assay Guidance Manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences, Bethesda, MD2021Google Scholar). This revealed slower on- and off-rates and a longer half-life for AM2/IMD–TAMRA (t1/2 ∼ 41 min) as compared with AM–TAMRA (t1/2 ∼ 2.6 min) (Table S2). The Kd values calculated from the on- and off-rates were ∼10-fold lower than the equilibrium Kd values. These discrepancies may reflect inaccuracies in fitting the kinetic data due to the signal decay issue and/or indicate that a single-step binding model does not appropriately describe these interactions. Next, we turned to dissociation kinetics experiments. The membranes were incubated with 10 nM of each probe for 25 min to reach equilibrium followed by injection of either 1 μM high-affinity unlabeled AM variant antagonist to initiate dissociation or buffer control. Signal decay was also evident in these experiments (Fig. S2H), but the decay could be corrected for by normalization to the buffer control injection. These data revealed much slower dissociation of AM2/IMD–TAMRA than AM–TAMRA and the dissociation curves were best fit by a two-phase exponential decay model (Fig. 2F). AM–TAMRA exhibited fast and slow component half-lives of 0.75 and 6.9 min, respectively, whereas AM2/IMD–TAMRA had fast and slow component half-lives of 7 and 76 min, respectively (Table S2). The fast components for AM–TAMRA and AM2/IMD–TAMRA accounted for 67% and 35% of the dissociation curves, respectively. These data are consistent with a two-step (un)binding process as proposed for other class B GPCR peptide ligands (40Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. et al.Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Crossref PubMed Scopus (436) Google Scholar, 41Hoare S.R. Mechanisms of peptide and nonpeptide ligand binding to class B G-protein-coupled receptors.Drug Discov. Today. 2005; 10: 417-427Crossref PubMed Scopus (309) Google Scholar, 42Castro M. Nikolaev V.O. Palm D. Lohse M.J. Vilardaga J.P. Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16084-16089Crossref PubMed Scopus (157) Google Scholar) and indicate that AM2/IMD–TAMRA has a substantially longer residence time than AM–TAMRA at the uncoupled AM2R. We also performed equilibrium binding and dissociation kinetics experiments in the presence of purified sumo-mini-Gs fusion protein, which is a G protein surrogate that locks the receptor in the active, coupled state (17Roehrkasse A.M. Warner M.L. Booe J.M. Pioszak A.A. Biochemical characterization of G protein coupling to calcitonin gene-related peptide and adrenomedullin receptors using a native PAGE assay.J. Biol. Chem. 2020; 295: 9736-9751Abstract Full Text Full Text PDF PubMed Sc

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