RESUMO
BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) is a potent vasodilator. While its signalling is assumed to be mediated via increases in cAMP, this study focused on elucidating the actual intracellular signalling pathways involved in CGRP-induced relaxation of human isolated coronary arteries (HCA). EXPERIMENTAL APPROACH: HCA were obtained from heart valve donors (27 M, 25 F, age 54 ± 2 years). Concentration-response curves to human α-CGRP or forskolin were constructed in HCA segments, incubated with different inhibitors of intracellular signalling pathways, and intracellular cAMP levels were measured with and without stimulation. RESULTS: Adenylyl cyclase (AC) inhibitors SQ22536 + DDA and MDL-12330A, and PKA inhibitors Rp-8-Br-cAMPs and H89, did not inhibit CGRP-induced relaxation of HCA, nor did the guanylyl cyclase inhibitor ODQ, PKG inhibitor KT5823, EPAC1/2 inhibitor ESI09, potassium channel blockers TRAM-34 + apamin, iberiotoxin or glibenclamide, or the Gαq inhibitor YM-254890. Phosphodiesterase inhibitors induced a concentration-dependent decrease in the response to KCl but did not potentiate relaxation to CGRP. Relaxation to forskolin was not blocked by PKA or AC inhibitors, although AC inhibitors significantly inhibited the increase in cAMP. Inhibition of Gßγ subunits using gallein significantly inhibited the relaxation to CGRP in human coronary arteries. CONCLUSION: While CGRP signalling is generally assumed to act via cAMP, the CGRP-induced vasodilation in HCA was not inhibited by targeting this intracellular signalling pathway at different levels. Instead, inhibition of Gßγ subunits did inhibit the relaxation to CGRP, suggesting a different mechanism of CGRP-induced relaxation than generally believed.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Vasos Coronários , AMP Cíclico , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Vasodilatação , Humanos , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Vasos Coronários/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Masculino , Pessoa de Meia-Idade , AMP Cíclico/metabolismo , Vasodilatação/efeitos dos fármacos , Feminino , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Transdução de Sinais/efeitos dos fármacos , Técnicas In Vitro , Vasodilatadores/farmacologiaRESUMO
G-protein-coupled receptors (GPCRs) are the largest family of membrane proteins, regulate a plethora of physiological responses and are the therapeutic target for 30-40% of clinically-prescribed drugs. They are integral membrane proteins deeply embedded in the plasma membrane where they activate intracellular signalling via coupling to G-proteins and ß-arrestin. GPCRs are in intimate association with the bilayer lipids and that lipid environment regulates the signalling functions of GPCRs. This complex lipid 'landscape' is both heterogeneous and dynamic. GPCR function is modulated by bulk membrane properties including membrane fluidity, microdomains, curvature, thickness and asymmetry but GPCRs are also regulated by specific lipid:GPCR binding, including cholesterol and anionic lipids. Understanding the molecular mechanisms whereby GPCR signalling is regulated by lipids is a very active area of research currently. A major advance in membrane protein research in recent years was the application of poly(styrene-co-maleic acid) (SMA) copolymers. These spontaneously generate SMA lipid particles (SMALPs) encapsulating membrane protein in a nano-scale disc of cell membrane, thereby removing the historical need for detergent and preserving lipid:GPCR interaction. The focus of this review is how GPCR-SMALPs are increasing our understanding of GPCR structure and function at the molecular level. Furthermore, an increasing number of 'second generation' SMA-like copolymers have been reported recently. These are reviewed from the context of increasing our understanding of GPCR molecular mechanisms. Moreover, their potential as a novel platform for downstream biophysical and structural analyses is assessed and looking ahead, the translational application of SMA-like copolymers to GPCR drug discovery programmes in the future is considered.
Assuntos
Receptores Acoplados a Proteínas G , Membrana Celular , Lipídeos/química , Proteínas de Membrana/químicaRESUMO
Signalling of the calcitonin-like receptor (CLR) is multifaceted, due to its interaction with receptor activity modifying proteins (RAMPs), and three endogenous peptide agonists. Previous studies have focused on the bias of G protein signalling mediated by the receptor and receptor internalisation of the CLR-RAMP complex has been assumed to follow the same pattern as other Class B1 G Protein-Coupled Receptors (GPCRs). Here we sought to measure desensitisation of the three CLR-RAMP complexes in response to the three peptide agonists, through the measurement of ß-arrestin recruitment and internalisation. We then delved further into the mechanism of desensitisation through modulation of ß-arrestin activity and the expression of GPCR kinases (GRKs), a key component of homologous GPCR desensitisation. First, we have shown that CLR-RAMP1 is capable of potently recruiting ß-arrestin1 and 2, subsequently undergoing rapid endocytosis, and that CLR-RAMP2 and -RAMP3 also utilise these pathways, although to a lesser extent. Following this we have shown that agonist-dependent internalisation of CLR is ß-arrestin dependent, but not required for full agonism. Overexpression of GRK2-6 was then found to decrease receptor signalling, due to an agonist-independent reduction in surface expression of the CLR-RAMP complex. These results represent the first systematic analysis of the importance of ß-arrestins and GRKs in CLR-RAMP signal transduction and pave the way for further investigation regarding other Class B1 GPCRs.
RESUMO
The ß2-adrenoceptor (ß2AR) is a well-established target in asthma and a prototypical G protein-coupled receptor for biophysical studies. Solubilization of membrane proteins has classically involved the use of detergents. However, the detergent environment differs from the native membrane environment and often destabilizes membrane proteins. Use of amphiphilic copolymers is a promising strategy to solubilize membrane proteins within their native lipid environment in the complete absence of detergents. Here we show the isolation of the ß2AR in the polymer diisobutylene maleic acid (DIBMA). We demonstrate that ß2AR remains functional in the DIBMA lipid particle and shows improved thermal stability compared with the n-dodecyl-ß-D-maltopyranoside detergent-solubilized ß2AR. This unique method of extracting ß2AR offers significant advantages over previous methods routinely employed such as the introduction of thermostabilizing mutations and the use of detergents, particularly for functional biophysical studies.
RESUMO
Membrane proteins are of fundamental importance to cellular processes and nano-encapsulation strategies that preserve their native lipid bilayer environment are particularly attractive for studying and exploiting these proteins. Poly(styrene-co-maleic acid) (SMA) and related polymers poly(styrene-co-(N-(3-N',N'-dimethylaminopropyl)maleimide)) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) have revolutionised the study of membrane proteins by spontaneously solubilising membrane proteins direct from cell membranes within nanoscale discs of native bilayer called SMA lipid particles (SMALPs), SMILPs and DIBMALPs respectively. This systematic study shows for the first time, that conformational changes of the encapsulated protein are dictated by the solubilising polymer. The photoactivation pathway of rhodopsin (Rho), a G-protein-coupled receptor (GPCR), comprises structurally-defined intermediates with characteristic absorbance spectra that revealed conformational restrictions with styrene-containing SMA and SMI, so that photoactivation proceeded only as far as metarhodopsin-I, absorbing at 478 nm, in a SMALP or SMILP. In contrast, full attainment of metarhodopsin-II, absorbing at 382 nm, was observed in a DIBMALP. Consequently, different intermediate states of Rho could be generated readily by simply employing different SMA-like polymers. Dynamic light-scattering and analytical ultracentrifugation revealed differences in size and thermostability between SMALP, SMILP and DIBMALP. Moreover, encapsulated Rho exhibited different stability in a SMALP, SMILP or DIBMALP. Overall, we establish that SMA, SMI and DIBMA constitute a 'toolkit' of solubilising polymers, so that selection of the appropriate solubilising polymer provides a spectrum of useful attributes for studying membrane proteins.
Assuntos
Proteínas de Membrana , Polímeros , Bicamadas Lipídicas , Maleatos , Poliestirenos , Rodopsina , EstirenoRESUMO
Agonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor. However, its physiological relevance is not always clear. Using primary human cells and gene editing techniques, we demonstrate endogenous agonist bias with physiological consequences for the calcitonin receptor-like receptor, CLR. By switching the receptor-activity modifying protein (RAMP) associated with CLR we can "re-route" the physiological pathways activated by endogenous agonists calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2). AM2 promotes calcium-mediated nitric oxide signalling whereas CGRP and AM show pro-proliferative effects in cardiovascular cells, thus providing a rationale for the expression of the three peptides. CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and their importance in endogenous GPCR signalling.
Assuntos
Adrenomedulina/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/fisiologia , Hormônios Peptídicos/fisiologia , Receptores Acoplados a Proteínas G/agonistas , Proteína Semelhante a Receptor de Calcitonina/fisiologia , Células Cultivadas , AMP Cíclico/metabolismo , Células Endoteliais/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Receptores de Adrenomedulina/agonistas , Receptores de Adrenomedulina/análise , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/fisiologiaRESUMO
The first intracellular loop (ICL1) of G protein-coupled receptors (GPCRs) has received little attention, although there is evidence that, with the 8th helix (H8), it is involved in early conformational changes following receptor activation as well as contacting the G protein ß subunit. In class B1 GPCRs, the distal part of ICL1 contains a conserved R12.48KLRCxR2.46b motif that extends into the base of the second transmembrane helix; this is weakly conserved as a [R/H]12.48KL[R/H] motif in class A GPCRs. In the current study, the role of ICL1 and H8 in signaling through cAMP, iCa2+ and ERK1/2 has been examined in two class B1 GPCRs, using mutagenesis and molecular dynamics. Mutations throughout ICL1 can either enhance or disrupt cAMP production by CGRP at the CGRP receptor. Alanine mutagenesis identified subtle differences with regard elevation of iCa2+, with the distal end of the loop being particularly sensitive. ERK1/2 activation displayed little sensitivity to ICL1 mutation. A broadly similar pattern was observed with the glucagon receptor, although there were differences in significance of individual residues. Extending the study revealed that at the CRF1 receptor, an insertion in ICL1 switched signaling bias between iCa2+ and cAMP. Molecular dynamics suggested that changes in ICL1 altered the conformation of ICL2 and the H8/TM7 junction (ICL4). For H8, alanine mutagenesis showed the importance of E3908.49b for all three signal transduction pathways, for the CGRP receptor, but mutations of other residues largely just altered ERK1/2 activation. Thus, ICL1 may modulate GPCR bias via interactions with ICL2, ICL4 and the Gß subunit.
Assuntos
Motivos de Aminoácidos/fisiologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/ultraestrutura , Receptores de Hormônio Liberador da Corticotropina/ultraestrutura , Receptores de Glucagon/ultraestrutura , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Proteína Semelhante a Receptor de Calcitonina/fisiologia , Proteína Semelhante a Receptor de Calcitonina/ultraestrutura , Sinalização do Cálcio , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Simulação de Dinâmica Molecular , Domínios Proteicos , Estrutura Terciária de Proteína , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Proteína 1 Modificadora da Atividade de Receptores/fisiologia , Proteína 1 Modificadora da Atividade de Receptores/ultraestrutura , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/fisiologia , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Hormônio Liberador da Corticotropina/fisiologia , Receptores Acoplados a Proteínas G , Receptores de Glucagon/metabolismo , Receptores de Glucagon/fisiologiaRESUMO
The fundamental importance of membrane proteins in cellular processes has driven a marked increase in the use of membrane mimetic approaches for studying and exploiting these proteins. Nano-encapsulation strategies which preserve the native lipid bilayer environment are particularly attractive. Consequently, the use of poly(styrene co-maleic acid) (SMA) has been widely adopted to solubilise proteins directly from cell membranes by spontaneously forming "SMA Lipid Particles" (SMALPs). G-protein-coupled receptors (GPCRs) are ubiquitous "chemical switches", are central to cell signalling throughout the evolutionary tree, form the largest family of membrane proteins in humans and are a major drug discovery target. GPCR-SMALPs that retain binding capability would be a versatile platform for a wide range of down-stream applications. Here, using the adenosine A2A receptor (A2AR) as an archetypical GPCR, we show for the first time the utility of fluorescence correlation spectroscopy (FCS) to characterise the binding capability of GPCRs following nano-encapsulation. Unbound fluorescent ligand CA200645 exhibited a monophasic autocorrelation curve (dwell time, τD = 68 ± 2 µs; diffusion coefficient, D = 287 ± 15 µm2 s-1). In the presence of A2AR-SMALP, bound ligand was also evident (τD = 625 ± 23 µs; D = 30 ± 4 µm2 s-1). Using a non-receptor control (ZipA-SMALP) plus competition binding confirmed that this slower component represented binding to the encapsulated A2AR. Consequently, the combination of GPCR-SMALP and FCS is an effective platform for the quantitative real-time characterisation of nano-encapsulated receptors, with single molecule sensitivity, that will have widespread utility for future exploitation of GPCR-SMALPs in general.
Assuntos
Ligantes , Maleatos/química , Receptores Acoplados a Proteínas G/metabolismo , Estireno/química , Materiais Biomiméticos , Fluorescência , Humanos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Ligação Proteica , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/metabolismo , Receptores Acoplados a Proteínas G/química , Imagem Individual de Molécula , Espectrometria de FluorescênciaRESUMO
The use of styrene maleic acid co-polymer (SMA) for membrane protein extraction and purification has grown in recent years. SMA inserts in the membrane and assembles into small discs of bilayer encircled by polymer, termed SMA lipid particles (SMALPs). This allows purification of membrane proteins whilst maintaining their lipid bilayer environment. SMALPs offer several improvements over conventional detergent approaches, however there are limitations, most notably a sensitivity to low pH and divalent cations. Recently it was shown that the aliphatic diisobutylene-maleic acid (DIBMA) copolymer, was also able to directly solubilise membranes forming DIBMALPs (DIBMA lipid particles), and that this polymer overcame some of the limitations of SMA. In this study the ability of DIBMA to solubilise and purify functional membrane proteins has been compared to SMA. It was found that DIBMA is able to solubilise several different membrane proteins from different expression systems, however for some proteins it gives a lower yield and lower degree of purity than SMA. DIBMA extracted G protein-coupled receptors retain ligand- and G protein-binding. DIBMALPS are larger than SMALPs and display a decreased sensitivity to magnesium. However the stability of DIBMALPs appears to be lower than SMALPs. The lower purity and lower stability are likely linked to the larger size of the DIBMALP particle. However, this also offers a potentially less rigid lipid environment which may be more amenable to protein dynamics. Therefore the optimal choice of polymer will depend on which features of a protein are to be investigated.
Assuntos
Bicamadas Lipídicas/isolamento & purificação , Maleatos/química , Proteínas de Membrana/isolamento & purificação , Poliestirenos/química , Alcenos/química , Bicamadas Lipídicas/química , Gotículas Lipídicas/química , Proteínas de Membrana/químicaRESUMO
The adenosine 2A receptor (A2AR), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A2AR-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A2AR-SMALP encapsulated native lipids. The fluorescence spectrum of the A2AR-SMALP showed a characteristic broad emission peak at 330 nm, produced by endogenous Trp residues. The inverse agonist ZM241385 caused 30% increase in fluorescence emission, unusually accompanied by a red-shift in the emission wavelength. The emission spectrum also showed sub-peaks at 321 nm, 335 nm and 350 nm, indicating that individual Trp inhabited different environments following ZM241385 addition. There was no effect of the agonist NECA on the A2AR-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp2466.48 in TM6 and Trp2687.33 at the extracellular face of TM7, causing transition to a more hydrophobic environment. The fluorescent moiety IAEDANS was site-specifically introduced at the intracellular end of TM6 (residue 2316.33) to report on the dynamic cytoplasmic face of the A2AR. The inverse agonist ZM241385 caused a concentration-dependent increase in fluorescence emission as the IAEDANS moved to a more hydrophobic environment, consistent with closing the G-protein binding crevice. NECA generated only 30% of the effect of ZM241385. This study provides insight into the SMALP environment; encapsulation supported constitutive activity of the A2AR and ZM241385-induced conformational transitions but the agonist NECA generated only small effects.
Assuntos
Lipídeos/química , Receptor A2A de Adenosina/química , Estireno/química , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Maleatos/química , Pichia/química , Conformação Proteica , Espectrometria de Fluorescência/métodos , Triazinas/farmacologia , Triazóis/farmacologia , Triptofano/químicaRESUMO
The class B G protein-coupled receptor (GPCR) calcitonin receptor (CTR) is a drug target for osteoporosis and diabetes. N-glycosylation of asparagine 130 in its extracellular domain (ECD) enhances calcitonin hormone affinity with the proximal GlcNAc residue mediating this effect through an unknown mechanism. Here, we present two crystal structures of salmon calcitonin-bound, GlcNAc-bearing CTR ECD at 1.78 and 2.85 Å resolutions and analyze the mechanism of the glycan effect. The N130 GlcNAc does not contact the hormone. Surprisingly, the structures are nearly identical to a structure of hormone-bound, N-glycan-free ECD, which suggested that the GlcNAc might affect CTR dynamics not observed in the static crystallographic snapshots. Hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations revealed that glycosylation stabilized a ß-sheet adjacent to the N130 GlcNAc and the N-terminal α-helix near the peptide-binding site while increasing flexibility of the peptide-binding site turret loop. These changes due to N-glycosylation increased the ligand on-rate and decreased its off-rate. The glycan effect extended to RAMP-CTR amylin receptor complexes and was also conserved in the related CGRP receptor. These results reveal that N-glycosylation can modulate GPCR function by altering receptor dynamics.
Assuntos
Conformação Proteica , Receptores da Calcitonina/química , Receptores da Calcitonina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Glicosilação , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Transdução de SinaisRESUMO
Receptor component protein (RCP) is a 148 amino acid intracellular peripheral membrane protein, previously identified as promoting the coupling of CGRP to cAMP production at the CGRP receptor, a heterodimer of calcitonin receptor like-receptor (CLR), a family B G protein-coupled receptor (GPCR) and receptor activity modifying protein 1 (RAMP1). We extend these observations to show that it selectively enhances CGRP receptor coupling to Gs but not Gq or pERK activation. At other family B GPCRs, it enhances cAMP production at the calcitonin, corticotrophin releasing factor type 1a and glucagon-like peptide type 2 receptors with their cognate ligands but not at the adrenomedullin type 1 (AM1), gastric inhibitory peptide and glucagon-like peptide type 1 receptors, all expressed in transfected HEK293S cells. However, there is also cell-line variability as RCP did not enhance cAMP production at the endogenous calcitonin receptor in HEK293T cells and it has previously been reported that it is active on the AM1 receptor expressed on NIH3T3 cells. RCP appears to behave as a positive allosteric modulator at coupling a number of family B GPCRs to Gs, albeit in a manner that is regulated by cell-specific factors. It may exert its effects at the interface between the 2nd intracellular loop of the GPCR and Gs, although there is likely to be some overlap between this location and that occupied by the C-terminus of RAMPs if they bind to the GPCRs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Membrana/metabolismo , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Adrenomedulina/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina , Proteína Semelhante a Receptor de Calcitonina/química , Proteína Semelhante a Receptor de Calcitonina/metabolismo , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Ligantes , Hormônios Peptídicos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques.
Assuntos
Receptores Acoplados a Proteínas G/biossíntese , Animais , Linhagem Celular , Clonagem Molecular , Drosophila melanogaster , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Expressão Gênica , Maleatos/química , Poliestirenos/química , Receptores Acoplados a Proteínas G/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , SolubilidadeRESUMO
We report the first case of nonimmune hydrops fetalis (NIHF) associated with a recessive, in-frame deletion of V205 in the G protein-coupled receptor, Calcitonin Receptor-Like Receptor (hCALCRL). Homozygosity results in fetal demise from hydrops fetalis, while heterozygosity in females is associated with spontaneous miscarriage and subfertility. Using molecular dynamic modeling and in vitro biochemical assays, we show that the hCLR(V205del) mutant results in misfolding of the first extracellular loop, reducing association with its requisite receptor chaperone, receptor activity modifying protein (RAMP), translocation to the plasma membrane and signaling. Using three independent genetic mouse models we establish that the adrenomedullin-CLR-RAMP2 axis is both necessary and sufficient for driving lymphatic vascular proliferation. Genetic ablation of either lymphatic endothelial Calcrl or nonendothelial Ramp2 leads to severe NIHF with embryonic demise and placental pathologies, similar to that observed in humans. Our results highlight a novel candidate gene for human congenital NIHF and provide structure-function insights of this signaling axis for human physiology.
Assuntos
Sequência de Aminoácidos , Proteína Semelhante a Receptor de Calcitonina , Anormalidades Craniofaciais , Hidropisia Fetal , Linfangiectasia Intestinal , Linfedema , Camundongos Transgênicos , Deleção de Sequência , Animais , Proteína Semelhante a Receptor de Calcitonina/genética , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/metabolismo , Anormalidades Craniofaciais/patologia , Modelos Animais de Doenças , Feminino , Células HEK293 , Heterozigoto , Homozigoto , Humanos , Hidropisia Fetal/genética , Hidropisia Fetal/metabolismo , Hidropisia Fetal/patologia , Linfangiectasia Intestinal/genética , Linfangiectasia Intestinal/metabolismo , Linfangiectasia Intestinal/patologia , Linfedema/genética , Linfedema/metabolismo , Linfedema/patologia , Masculino , Camundongos , Placenta , GravidezRESUMO
Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). How CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor remains unclear. In this study, a photoaffinity cross-linker, p-azido l-phenylalanine (azF), was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimized to ensure efficient photolysis of azF residues near the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of ultraviolet-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the fifth transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu12 and Ala13 of CGRP, and Leu16 of CGRP is at the ECL/extracellular domain boundary of CLR. The reduced potency, Emax, and affinity of [Leu16Ala]-human α CGRP are consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/química , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína Semelhante a Receptor de Calcitonina/química , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Proteína 1 Modificadora da Atividade de Receptores/química , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/genética , Proteína Semelhante a Receptor de Calcitonina/genética , Humanos , Mutagênese , Ligação Proteica , Estrutura Secundária de Proteína , Proteína 1 Modificadora da Atividade de Receptores/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of these challenging targets. This review focuses on the microbial expression systems that are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. We provide an overview of the host strains, tags and promoters that, in our experience, are most likely to yield protein suitable for structural and functional characterization. We also catalogue the detergents used for solubilization and crystallization studies of these proteins. Here, we emphasize a combination of practical methods, not necessarily high-throughput, which can be implemented in any laboratory equipped for recombinant DNA technology and microbial cell culture.
Assuntos
Bactérias/genética , Proteínas de Membrana/biossíntese , Proteínas Recombinantes/biossíntese , Leveduras/genética , Plasmídeos , Regiões Promotoras GenéticasRESUMO
The calcitonin/CGRP family of peptides includes calcitonin, α and ß CGRP, amylin, adrenomedullin (AM) and adrenomedullin 2/intermedin (AM2/IMD). Their receptors consist of one of two GPCRs, the calcitonin receptor (CTR) or the calcitonin receptor-like receptor (CLR). Further diversity arises from heterodimerization of these GPCRs with one of three receptor activity-modifying proteins (RAMPs). This gives the CGRP receptor (CLR/RAMP1), the AM1 and AM2 receptors (CLR/RAMP2 or RAMP3) and the AMY1, AMY2 and AMY3 receptors (CTR/RAMPs1-3 complexes, respectively). Apart from the CGRP receptor, there are only peptide antagonists widely available for these receptors, and these have limited selectivity, thus defining the function of each receptor in vivo remains challenging. Further challenges arise from the probable co-expression of CTR with the CTR/RAMP complexes and species-dependent splice variants of the CTR (CT(a) and CT(b) ). Furthermore, the AMY1(a) receptor is activated equally well by both amylin and CGRP, and the preferred receptor for AM2/IMD has been unclear. However, there are clear therapeutic rationales for developing agents against the various receptors for these peptides. For example, many agents targeting the CGRP system are in clinical trials, and pramlintide, an amylin analogue, is an approved therapy for insulin-requiring diabetes. This review provides an update on the pharmacology of the calcitonin family of peptides by members of the corresponding subcommittee of the International Union of Basic and Clinical Pharmacology and colleagues.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Calcitonina/metabolismo , Calcitonina/farmacologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Animais , Calcitonina/química , Peptídeo Relacionado com Gene de Calcitonina/química , Proteína Semelhante a Receptor de Calcitonina/química , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Humanos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Receptores da Calcitonina/química , Receptores da Calcitonina/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/químicaRESUMO
Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors are heteromers of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, and one of three receptor activity-modifying proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP production, modeling the active state conformations of CGRP and AM receptors in complex with the Gs C-terminus and conducting molecular dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on receptor signaling were seen with H295A5.40b, I298A5.43b, L302A5.47b, N305A5.50b, L345A6.49b and E348A6.52b, F349A6.53b and H374A7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H3747.47b to alanine increased AM potency 100-fold in the CGRP receptor. The molecular dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Proteínas Modificadoras da Atividade de Receptores/metabolismo , Receptores de Adrenomedulina/metabolismo , Animais , Células COS , Peptídeo Relacionado com Gene de Calcitonina/química , Peptídeo Relacionado com Gene de Calcitonina/genética , Proteína Semelhante a Receptor de Calcitonina/química , Proteína Semelhante a Receptor de Calcitonina/genética , Chlorocebus aethiops , AMP Cíclico/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Ensaio Radioligante , Proteínas Modificadoras da Atividade de Receptores/química , Proteínas Modificadoras da Atividade de Receptores/genética , Receptores de Adrenomedulina/química , Receptores de Adrenomedulina/genética , Proteínas Recombinantes de Fusão , TransfecçãoRESUMO
The second extracellular loop (ECL2) of the G protein-coupled receptor (GPCR) family is important for ligand interaction and drug discovery. ECL2 of the family B cardioprotective calcitonin gene-related peptide (CGRP) receptor is required for cell signaling. Family B GPCR ligands have two regions; the N-terminus mediates receptor activation, and the remainder confers high-affinity binding. Comparing antagonism of CGRP8-37 at a number of point mutations of ECL2 of the CGRP receptor, we show that the ECL2 potentially facilitates interaction with up to the 18 N-terminal residues of CGRP. This has implications for understanding family B GPCR activation and for drug design at the CGRP receptor.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Proteína Semelhante a Receptor de Calcitonina/agonistas , Mióticos/farmacologia , Modelos Moleculares , Fragmentos de Peptídeos/farmacologia , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/agonistas , Transdução de Sinais/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , 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/química , Proteína Semelhante a Receptor de Calcitonina/genética , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Chlorocebus aethiops , Cinética , Ligantes , Mióticos/química , Mióticos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Proteína 1 Modificadora da Atividade de Receptores/química , Proteína 1 Modificadora da Atividade de Receptores/genética , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/química , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/genética , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Homologia Estrutural de ProteínaRESUMO
The extracellular loop 2 (ECL2) region is the most conserved of the three ECL domains in family B G protein-coupled receptors (GPCRs) and has a fundamental role in ligand binding and activation across the receptor super-family. ECL2 is fundamental for ligand-induced activation of the calcitonin gene related peptide (CGRP) receptor, a family B GPCR implicated in migraine and heart disease. In this study we apply a comprehensive targeted non-alanine substitution analysis method and molecular modelling to the functionally important residues of ECL2 to reveal key molecular interactions. We identified an interaction network between R274/Y278/D280/W283. These amino acids had the biggest reduction in signalling following alanine substitution analysis and comprise a group of basic, acidic and aromatic residues conserved in the wider calcitonin family of class B GPCRs. This study identifies key and varied constraints at each locus, including diverse biochemical requirements for neighbouring tyrosine residues and a W283H substitution that recovered wild-type (WT) signalling, despite the strictly conserved nature of the central ECL2 tryptophan and the catastrophic effects on signalling of W283A substitution. In contrast, while the distal end of ECL2 requires strict conservation of hydrophobicity or polarity in each position, mutation of these residues never has a large effect. This approach has revealed linked networks of amino acids, consistent with structural models of ECL2 and likely to represent a shared structural framework at an important ligand-receptor interface that is present across the family B GPCRs.