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.
Assuntos
Adrenomedulina , Peptídeo Relacionado com Gene de Calcitonina , Proteínas Modificadoras da Atividade de Receptores , Receptores de Adrenomedulina , Receptores Acoplados a Proteínas G , Animais , Humanos , Adrenomedulina/química , Adrenomedulina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína Semelhante a Receptor de Calcitonina/genética , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Chlorocebus aethiops , Células COS , AMP Cíclico/metabolismo , Células HEK293 , Modelos Moleculares , Simulação de Dinâmica Molecular , Estabilidade Proteica , Proteínas Modificadoras da Atividade de Receptores/química , Proteínas Modificadoras da Atividade de Receptores/genética , Proteínas Modificadoras da Atividade de Receptores/metabolismo , Receptores de Adrenomedulina/genética , Receptores de Adrenomedulina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
Apyrase from potato (Solanum tuberosum) is a divalent metal ion-dependent enzyme that catalyzes the hydrolysis of nucleoside di- and tri-phosphates with broad substrate specificity. The enzyme is widely used to manipulate nucleotide levels such as in the G protein-coupled receptor (GPCR) field where it is used to deplete guanine nucleotides to stabilize nucleotide-free ternary agonist-GPCR-G protein complexes. Potato apyrase is available commercially as the native enzyme purified from potatoes or as a recombinant protein, but these are prohibitively expensive for some research applications. Here, we report a relatively simple method for the bacterial production of soluble, active potato apyrase. Apyrase has several disulfide bonds, so we co-expressed the enzyme bearing a C-terminal (His)6 tag with the E. coli disulfide isomerase DsbC at low temperature (18 °C) in the oxidizing cytoplasm of E. coli Origami B (DE3). This allowed low level production of soluble apyrase. A two-step purification procedure involving Ni-affinity followed by Cibacron Blue-affinity chromatography yielded highly purified apyrase at a level of â¼0.5 mg per L of bacterial culture. The purified enzyme was functional for ATP hydrolysis in an ATPase assay and for GTP/GDP hydrolysis in a GPCR-G protein coupling assay. This methodology enables the time- and cost-efficient production of recombinant apyrase for various research applications.
Assuntos
Apirase , Solanum tuberosum , Apirase/genética , Apirase/química , Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Proteínas Recombinantes/química , Solanum tuberosum/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a ß-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.
Assuntos
Proteína Semelhante a Receptor de Calcitonina/química , Peptídeos/química , Proteína 1 Modificadora da Atividade de Receptores/química , Proteína 2 Modificadora da Atividade de Receptores/química , Adrenomedulina/química , Adrenomedulina/genética , Adrenomedulina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Células COS , Peptídeo Relacionado com Gene de Calcitonina/química , Peptídeo Relacionado com Gene de Calcitonina/genética , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína Semelhante a Receptor de Calcitonina/genética , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Chlorocebus aethiops , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteína 1 Modificadora da Atividade de Receptores/genética , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Proteína 2 Modificadora da Atividade de Receptores/genética , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) have overlapping and unique functions in the nervous and circulatory systems including vasodilation, cardioprotection, and pain transmission. Their actions are mediated by the class B calcitonin-like G protein-coupled receptor (CLR), which heterodimerizes with three receptor activity-modifying proteins (RAMP1-3) that determine its peptide ligand selectivity. How the three agonists and RAMPs modulate CLR binding to transducer proteins remains poorly understood. Here, we biochemically characterized agonist-promoted G protein coupling to each CLR·RAMP complex. We adapted a native PAGE method to assess the formation and thermostabilities of detergent-solubilized fluorescent protein-tagged CLR·RAMP complexes expressed in mammalian cells. Addition of agonist and the purified Gs protein surrogate mini-Gs (mGs) yielded a mobility-shifted agonist·CLR·RAMP·mGs quaternary complex gel band that was sensitive to antagonists. Measuring the apparent affinities of the agonists for the mGs-coupled receptors and of mGs for the agonist-occupied receptors revealed that both ligand and RAMP control mGs coupling and defined how agonist engagement of the CLR extracellular and transmembrane domains affects transducer recruitment. Using mini-Gsq and -Gsi chimeras, we observed a coupling rank order of mGs > mGsq > mGsi for each receptor. Last, we demonstrated the physiological relevance of the native gel assays by showing that they can predict the cAMP-signaling potencies of AM and AM2/IMD chimeras. These results highlight the power of the native PAGE assay for membrane protein biochemistry and provide a biochemical foundation for understanding the molecular basis of shared and distinct signaling properties of CGRP, AM, and AM2/IMD.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Eletroforese em Gel de Poliacrilamida Nativa , Receptores de Adrenomedulina , Animais , Células COS , Peptídeo Relacionado com Gene de Calcitonina/química , Peptídeo Relacionado com Gene de Calcitonina/genética , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Chlorocebus aethiops , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Domínios Proteicos , Receptores de Adrenomedulina/química , Receptores de Adrenomedulina/genética , Receptores de Adrenomedulina/metabolismo , Sistemas do Segundo MensageiroRESUMO
The cardioprotective vasodilator peptide adrenomedullin 2/intermedin (AM2/IMD) and the related adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) signal through three heterodimeric receptors comprising the calcitonin receptor-like class B G protein-coupled receptor (CLR) and a variable receptor activity-modifying protein (RAMP1, -2, or -3) that determines ligand selectivity. The CGRP receptor (RAMP1:CLR) favors CGRP binding, whereas the AM1 (RAMP2:CLR) and AM2 (RAMP3:CLR) receptors favor AM binding. How AM2/IMD binds the receptors and how RAMPs modulate its binding is unknown. Here, we show that AM2/IMD binds the three purified RAMP-CLR extracellular domain (ECD) complexes with a selectivity profile that is distinct from those of CGRP and AM. AM2/IMD bound all three ECD complexes but preferred the CGRP and AM2 receptor complexes. A 2.05 Å resolution crystal structure of an AM2/IMD antagonist fragment-bound RAMP1-CLR ECD complex revealed that AM2/IMD binds the complex through a unique triple ß-turn conformation that was confirmed by peptide and receptor mutagenesis. Comparisons of the receptor-bound conformations of AM2/IMD, AM, and a high-affinity CGRP analog revealed differences that may have implications for biased signaling. Guided by the structure, enhanced-affinity AM2/IMD antagonist variants were developed, including one that discriminates the AM1 and AM2 receptors with â¼40-fold difference in affinities and one stabilized by an intramolecular disulfide bond. These results reveal differences in how the three peptides engage the receptors, inform development of AM2/IMD-based pharmacological tools and therapeutics, and provide insights into RAMP modulation of receptor pharmacology.
Assuntos
Adrenomedulina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Hormônios Peptídicos/metabolismo , Proteínas Modificadoras da Atividade de Receptores/metabolismo , Receptores de Adrenomedulina/metabolismo , Adrenomedulina/isolamento & purificação , Peptídeo Relacionado com Gene de Calcitonina/isolamento & purificação , Proteína Semelhante a Receptor de Calcitonina/isolamento & purificação , Desenho de Fármacos , Células HEK293 , Humanos , Ligantes , Mutagênese Sítio-Dirigida , Hormônios Peptídicos/antagonistas & inibidores , Hormônios Peptídicos/genética , Hormônios Peptídicos/isolamento & purificação , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Proteína 1 Modificadora da Atividade de Receptores/isolamento & purificação , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Proteína 2 Modificadora da Atividade de Receptores/isolamento & purificação , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Proteína 3 Modificadora da Atividade de Receptores/isolamento & purificação , Proteína 3 Modificadora da Atividade de Receptores/metabolismo , Proteínas Modificadoras da Atividade de Receptores/isolamento & purificação , Receptores de Adrenomedulina/isolamento & purificaçãoRESUMO
It is now recognized that G protein-coupled receptors (GPCRs), once considered largely independent functional units, have a far more diverse molecular architecture. Receptor activity-modifying proteins (RAMPs) provide an important example of proteins that interact with GPCRs to modify their function. RAMPs are able to act as pharmacological switches and chaperones, and they can regulate signaling and/or trafficking in a receptor-dependent manner. This review covers recent discoveries in the RAMP field and summarizes the known GPCR partners and functions of RAMPs. We also discuss the first peptide-bound structures of RAMP-GPCR complexes, which give insight into the molecular mechanisms that enable RAMPs to alter the pharmacology and signaling of GPCRs.
Assuntos
Proteínas Modificadoras da Atividade de Receptores/metabolismo , Animais , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Binding of the vasodilator peptides adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) to the class B G protein-coupled receptor calcitonin receptor-like receptor (CLR) is modulated by receptor activity-modifying proteins (RAMPs). RAMP1 favors CGRP, whereas RAMP2 and RAMP3 favor AM. Crystal structures of peptide-bound RAMP1/2-CLR extracellular domain (ECD) heterodimers suggested RAMPs alter ligand preference through direct peptide contacts and allosteric modulation of CLR. Here, we probed this dual mechanism through rational structure-guided design of AM and CGRP antagonist variants. Variants were characterized for binding to purified RAMP1/2-CLR ECD and for antagonism of the full-length CGRP (RAMP1:CLR), AM1 (RAMP2:CLR), and AM2 (RAMP3:CLR) receptors. Short nanomolar affinity AM(37-52) and CGRP(27-37) variants were obtained through substitutions including AM S45W/Q50W and CGRP K35W/A36S designed to stabilize their ß-turn. K46L and Y52F substitutions designed to exploit RAMP allosteric effects and direct peptide contacts, respectively, yielded AM variants with selectivity for the CGRP receptor over the AM1 receptor. AM(37-52) S45W/K46L/Q50W/Y52F exhibited nanomolar potency at the CGRP receptor and micromolar potency at AM1 A 2.8-Å resolution crystal structure of this variant bound to the RAMP1-CLR ECD confirmed that it bound as designed. CGRP(27-37) N31D/S34P/K35W/A36S exhibited potency and selectivity comparable to the traditional antagonist CGRP(8-37). Giving this variant the ability to contact RAMP2 through the F37Y substitution increased affinity for AM1, but it still preferred the CGRP receptor. These potent peptide antagonists with altered selectivity inform the development of AM/CGRP-based pharmacological tools and support the hypothesis that RAMPs alter CLR ligand selectivity through allosteric effects and direct peptide contacts.
Assuntos
Adrenomedulina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/antagonistas & inibidores , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Desenho de Fármacos , Receptores Acoplados a Proteínas G/metabolismo , Adrenomedulina/genética , Sequência de Aminoácidos , Animais , Células COS , Peptídeo Relacionado com Gene de Calcitonina/genética , Chlorocebus aethiops , Humanos , Ligantes , Estrutura Secundária de Proteína , Proteína 1 Modificadora da Atividade de Receptores/antagonistas & inibidores , Proteína 1 Modificadora da Atividade de Receptores/genética , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Proteína 2 Modificadora da Atividade de Receptores/antagonistas & inibidores , Proteína 2 Modificadora da Atividade de Receptores/genética , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Proteína 3 Modificadora da Atividade de Receptores/antagonistas & inibidores , Proteína 3 Modificadora da Atividade de Receptores/genética , Proteína 3 Modificadora da Atividade de Receptores/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMO
The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited â¼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI- cells, which yield core N-glycans (Man5GlcNAc2). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity â¼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N â Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.
Assuntos
Asparagina/metabolismo , Calcitonina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Receptores da Calcitonina/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Substituição de Aminoácidos , Asparagina/química , Sítios de Ligação , Calcitonina/química , Glicosilação , Células HEK293 , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Cinética , Ligantes , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Conformação Molecular , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteína 2 Modificadora da Atividade de Receptores/agonistas , Proteína 2 Modificadora da Atividade de Receptores/química , Proteína 2 Modificadora da Atividade de Receptores/genética , Receptores da Calcitonina/agonistas , Receptores da Calcitonina/química , Receptores da Calcitonina/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity.
Assuntos
Peptídeos/metabolismo , Receptores da Calcitonina/metabolismo , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Regulação Alostérica/fisiologia , Motivos de Aminoácidos , Células HEK293 , Humanos , Peptídeos/química , Peptídeos/genética , Estrutura Terciária de Proteína , Proteína 1 Modificadora da Atividade de Receptores/química , Proteína 1 Modificadora da Atividade de Receptores/genética , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Proteína 2 Modificadora da Atividade de Receptores/química , Proteína 2 Modificadora da Atividade de Receptores/genética , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Receptores da Calcitonina/química , Receptores da Calcitonina/genética , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/química , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genéticaRESUMO
Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.
Assuntos
Adrenomedulina/metabolismo , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Proteína 3 Modificadora da Atividade de Receptores/metabolismo , Adrenomedulina/genética , Sequência de Aminoácidos , Proteína Semelhante a Receptor de Calcitonina/química , Proteína Semelhante a Receptor de Calcitonina/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Proteína 2 Modificadora da Atividade de Receptores/química , Proteína 2 Modificadora da Atividade de Receptores/genética , Proteína 3 Modificadora da Atividade de Receptores/química , Proteína 3 Modificadora da Atividade de Receptores/genética , Receptores de Adrenomedulina/química , Receptores de Adrenomedulina/genética , Receptores de Adrenomedulina/metabolismo , Alinhamento de SequênciaRESUMO
Secreted R-spondin proteins (RSPOs1-4) function as adult stem cell growth factors by potentiating Wnt signaling. Simultaneous binding of distinct regions of the RSPO Fu1-Fu2 domain module to the extracellular domains (ECDs) of the LGR4 G protein-coupled receptor and the ZNRF3 transmembrane E3 ubiquitin ligase regulates Wnt receptor availability. Here, we examine the molecular basis for the differing signaling strengths of RSPOs1-4 using purified RSPO Fu1-Fu2, LGR4 ECD, and ZNRF3 ECD proteins in Wnt signaling and receptor binding assays, and we engineer novel high-potency RSPOs. RSPO2/3/4 had similar signaling potencies that were stronger than that of RSPO1, whereas RSPO1/2/3 had similar efficacies that were greater than that of RSPO4. The RSPOs bound LGR4 with affinity rank order RSPO4 > RSPO2/3 > RSPO1 and ZNRF3 with affinity rank order RSPO2/3 > > RSPO1 > RSPO4. An RSPO2-4 chimera combining RSPO2 ZNRF3 binding with RSPO4 LGR4 binding was a "Superspondin" that exhibited enhanced ternary complex formation and 10-fold stronger signaling potency than RSPO2 and efficacy equivalent to RSPO2. An RSPO4-1 chimera combining RSPO4 ZNRF3 binding with RSPO1 LGR4 binding was a "Poorspondin" that exhibited signaling potency similar to RSPO1 and efficacy equivalent to RSPO4. Conferring increased ZNRF3 binding upon RSPO4 with amino acid substitutions L56F, I58L, and I63M enhanced its signaling potency and efficacy. Our results reveal the molecular basis for RSPOs1-4 activity differences and suggest that signaling potency is determined by ternary complex formation ability, whereas efficacy depends on ZNRF3 recruitment. High-potency RSPOs may be of value for regenerative medicine and/or therapeutic applications.
Assuntos
Células-Tronco Adultas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Engenharia de Proteínas/métodos , Trombospondinas/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Trombospondinas/químicaRESUMO
Calcitonin gene-related peptides alpha and beta (αCGRP, ßCGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) function in pain signaling, neuroimmune communication, and regulation of the cardiovascular and lymphatic systems by activating either of two class B GPCRs, CLR and CTR, in complex with a RAMP1, -2, or -3 modulatory subunit. Inspired by our recent discovery that AM2/IMD(1-47) activation of CLR-RAMP3 elicits long duration cAMP signaling, here we used a live-cell cAMP biosensor assay to characterize the signaling kinetics of the two CGRP peptides and several bioactive AM and AM2/IMD fragments with variable N-terminal extensions. Remarkably, AM2/IMD(8-47) and AM2/IMD-53 exhibited even longer duration signaling than the 1-47 fragment. AM2/IMD(8-47) was a striking 8-fold longer acting than AM(13-52) at CLR-RAMP3. In contrast, the N-terminal extension of AM had no effect on signaling duration. AM(1-52) and (13-52) were equally short-acting. Analysis of AM2/IMD-AM mid-region chimeras and AM2/IMD R23 and R33 point mutants showed the importance of these residues for long-duration signaling and identified AM2/IMD peptides that exhibited up to 17-fold diminished signaling duration at CLR-RAMP3, while retaining near wildtype signaling potencies. ßCGRP was â¼ 3-fold longer acting than αCGRP at the CGRP (CLR-RAMP1) and the amylin1 (CTR-RAMP1) receptors. Chimeric CGRP peptides showed that the single residue difference near the N-terminus, and the two differences in the mid-region, equally contributed to the longer duration of ßCGRP signaling. This work uncovers key temporal differences in cAMP signaling among the CGRP family peptides, elucidates the structural bases thereof, and provides pharmacological tools for studying long-duration AM2/IMD signaling.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Transdução de Sinais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/genética , Peptídeo Relacionado com Gene de Calcitonina/química , Humanos , Transdução de Sinais/fisiologia , Células HEK293 , AMP Cíclico/metabolismo , Adrenomedulina/metabolismo , Adrenomedulina/química , Adrenomedulina/genética , Sequência de AminoácidosRESUMO
R-Spondins are secreted glycoproteins (RSPO1-RSPO4) that have proliferative effects on adult stem cells by potentiating Wnt signaling. RSPO actions are mediated by the leucine-rich repeat (LRR)-containing seven-transmembrane receptors LGR4-LGR6 and the transmembrane E3 ubiquitin ligases ZNRF3 and RNF43. Here, we present a methodology for the bacterial expression and purification of the signaling competent, cysteine-rich Fu1-Fu2 domains of the four human RSPOs, a fragment of the human LGR4 extracellular domain (ECD) containing LRR1-14, and the human ZNRF3 ECD. In a cell-based signaling assay, the nonglycosylated RSPOs enhanced low-dose Wnt3a signaling with potencies comparable to those of mammalian cell-produced RSPOs and RSPO2 and -3 were more potent than RSPO1 and -4. LGR4 LRR1-14 and ZNRF3 ECD inhibited RSPO2-enhanced Wnt3a signaling. The RSPOs bound LGR4 LRR1-14 with nanomolar affinities that decreased in the following order in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay: RSPO4 > RSPO2 > RSPO3 > RSPO1. RSPO-receptor interactions were further characterized with a native gel electrophoretic mobility shift assay, which corroborated the RSPO-LGR4 TR-FRET results and indicated that RSPOs weakly bound ZNRF3 with affinities that decreased in the following order: RSPO2 > RSPO3 > RSPO1. RSPO4:ZNRF3 complexes were not detected. Lastly, ternary RSPO:LGR4:ZNRF3 complexes were detected for RSPO2 and -3. Our results indicate that RSPO and LGR4 N-glycans are dispensable for function, demonstrate RSPO-mediated ternary complex formation, and suggest that the stronger signaling potencies of RSPO2 and -3 result from their strong binding of both receptors. Our unique protein production methodology may provide a cost-effective source of recombinant RSPOs for regenerative medicine applications.
Assuntos
Células-Tronco Adultas/metabolismo , Escherichia coli/genética , Expressão Gênica , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Escherichia coli/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Proteína Wnt3/metabolismoRESUMO
Adrenomedullin (AM) is a peptide hormone that is a potent vasodilator and is essential for vascular development. The AM receptor is a heterodimeric cell surface receptor composed of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, in association with either of two receptor activity modifying protein (RAMP) coreceptors, RAMP2 or -3. The extracellular domains (ECDs) of CLR and the RAMPs form the primary AM binding site. Here, we present novel methodology for expression and purification of a heterodimeric AM receptor ECD complex as an MBP-CLR ECD fusion protein in association with the RAMP2 ECD. Co-expression of the RAMP2 ECD with the disulfide bond isomerase DsbC in the oxidizing cytoplasm of E. coli trxB gor enabled proper disulfide formation in vivo. The isolated RAMP2 ECD was purified to homogeneity. Co-expression of a soluble MBP-CLR ECD fusion protein with DsbC in E. coli trxB gor yielded a heterogeneous mixture of species with misfolded ECD. Incubation of affinity-purified MBP-CLR ECD in vitro with purified RAMP2 ECD, DsbC, and glutathione redox buffer promoted proper folding of the CLR ECD and formation of a stable MBP-CLR ECD:RAMP2 ECD complex that was purified by size-exclusion chromatography and which exhibited specific AM binding. Approximately 40mg of highly purified complex was obtained starting with 6L bacterial cultures for each protein. The methodology reported here will facilitate structure/function studies of the AM receptor.
Assuntos
Proteína Semelhante a Receptor de Calcitonina , Complexos Multiproteicos/isolamento & purificação , Receptores de Adrenomedulina/genética , Receptores de Adrenomedulina/isolamento & purificação , Sequência de Aminoácidos , Sítios de Ligação , Proteína Semelhante a Receptor de Calcitonina/química , Proteína Semelhante a Receptor de Calcitonina/isolamento & purificação , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Dissulfetos/química , Humanos , Complexos Multiproteicos/química , Ligação Proteica , Estrutura Terciária de Proteína/genética , Proteína 1 Modificadora da Atividade de Receptores , Proteína 2 Modificadora da Atividade de Receptores/química , Proteína 2 Modificadora da Atividade de Receptores/isolamento & purificação , Proteína 3 Modificadora da Atividade de Receptores/química , Proteína 3 Modificadora da Atividade de Receptores/isolamento & purificação , Receptores de Adrenomedulina/biossíntese , Receptores de Adrenomedulina/química , Transdução de Sinais , Vasodilatadores/química , Vasodilatadores/isolamento & purificaçãoRESUMO
The signaling peptides adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and CGRP have overlapping and distinct functions in the cardiovascular, lymphatic, and nervous systems by activating three shared receptors comprised of the class B GPCR CLR in complex with a RAMP1, -2, or -3 modulatory subunit. Here, we report that AM2/IMD, which is thought to be a non-selective agonist, is kinetically selective for CLR-RAMP3, known as the AM 2 R. AM2/IMD-AM 2 R elicited substantially longer duration cAMP signaling than the eight other peptide-receptor combinations due to AM2/IMD slow off-rate binding kinetics. The regions responsible for the slow off-rate were mapped to the AM2/IMD mid-region and the RAMP3 extracellular domain. MD simulations revealed how these bestow enhanced stability to the complex. Our results uncover AM2/IMD-AM 2 R as a cognate pair with unique temporal features, define the mechanism of kinetic selectivity, and explain how AM2/IMD and RAMP3 collaborate to shape the signaling output of a clinically important GPCR.
RESUMO
The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2α isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2α ECD bound to each of the Ucn peptides. The CRFR2α ECD forms the same fold observed for the CRFR1 and mouse CRFR2ß ECDs but contains a unique N-terminal α-helix formed by its pseudo signal peptide. The CRFR2α ECD peptide-binding site architecture is similar to that of CRFR1, and binding of the α-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2α Pro-100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu-104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala-35 is incompatible with the negatively charged Glu-104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.
Assuntos
Receptores de Hormônio Liberador da Corticotropina/química , Animais , Sítios de Ligação , Hormônio Liberador da Corticotropina/química , Hormônio Liberador da Corticotropina/genética , Hormônio Liberador da Corticotropina/metabolismo , Cristalografia por Raios X , Humanos , Camundongos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Receptores de Hormônio Liberador da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Urocortinas/química , Urocortinas/genética , Urocortinas/metabolismoRESUMO
The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an alpha-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.
Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Cristalografia por Raios X , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Técnicas In Vitro , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Hormônio Paratireóideo/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , TransfecçãoRESUMO
Parathyroid hormone (PTH) is central to calcium homeostasis and bone maintenance in vertebrates, and as such it has been used for treating osteoporosis. It acts primarily by binding to its receptor, PTH1R, a member of the class B G protein-coupled receptor (GPCR) family that also includes receptors for glucagon, calcitonin, and other therapeutically important peptide hormones. Despite considerable interest and much research, determining the structure of the receptor-hormone complex has been hindered by difficulties in purifying the receptor and obtaining diffraction-quality crystals. Here, we present a method for expression and purification of the extracellular domain (ECD) of human PTH1R engineered as a maltose-binding protein (MBP) fusion that readily crystallizes. The 1.95-A structure of PTH bound to the MBP-PTH1R-ECD fusion reveals that PTH docks as an amphipathic helix into a central hydrophobic groove formed by a three-layer alpha-beta-betaalpha fold of the PTH1R ECD, resembling a hot dog in a bun. Conservation in the ECD scaffold and the helical structure of peptide hormones emphasizes this hot dog model as a general mechanism of hormone recognition common to class B GPCRs. Our findings reveal critical insights into PTH actions and provide a rational template for drug design that targets this hormone signaling pathway.
Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Sequência Conservada , Cristalografia por Raios X , Expressão Gênica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Hormônio Paratireóideo/química , Hormônio Paratireóideo/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/isolamento & purificação , Sensibilidade e Especificidade , Alinhamento de SequênciaRESUMO
G protein-coupled receptors (GPCRs) are a large family of membrane-embedded receptors that have diverse roles in physiology and are major drug targets. GPCRs transduce an agonist binding signal across the membrane to activate intracellular heterotrimeric G proteins. The dynamic nature of the receptors and the complexity of their interactions with agonists and G proteins present significant challenges for biochemical studies. Most biochemical/biophysical methods that have been employed to study GPCR-G protein coupling require purified receptors and are technically difficult. Here, we provide a protocol for a relatively simple and time- and cost-effective membrane protein native PAGE assay, to visualize and biochemically characterize agonist-dependent coupling of detergent-solubilized GPCRs to purified G protein surrogate "mini-G" proteins, which stabilize the receptor in an active state. The assay was developed for our studies of the calcitonin receptor-like receptor, a class B GPCR that mediates the actions of calcitonin gene-related peptide and adrenomedullin peptide agonists. It does not require a purified receptor and it can be used in a screening format with transiently-transfected adherent mammalian cell cultures, to quickly identify detergent-stable complexes amenable to study, or in a quantitative format with membrane preparations, to determine apparent affinities of agonists for the mini-G-coupled receptor and apparent affinities of mini-G proteins for the agonist-occupied receptor. The latter provides a partial measure of agonist efficacy. The method should be applicable to other GPCRs, and has the potential to be adapted to the study of other challenging membrane proteins and their complexes with binding partners. Graphic abstract: Visualizing agonist-dependent mini-G protein coupling and determining apparent binding affinities using the native PAGE assay quantitative formats.