RESUMO
Mutations within the "X1BBX2X3B" motif or its variants in the junction of the third intracellular (i3) loop and the sixth transmembrane domain (TM6) have been shown to lead to constitutive activation of several G protein-coupled receptors (GPCRs). In this study, T6.34(279) at the X3 locus of the rat mu opioid receptor was mutated to Lys and Asp, and the mutants were examined for binding and signaling properties. The T6.34(279)K mutant was poorly expressed, and pretreatment with naloxone greatly enhanced its expression. This construct exhibited properties identified previously with constitutive activation: (1) compared with the wild type, it produced much higher agonist-independent [35S]GTPgammaS binding, which was abolished by pertussis toxin treatment; (2) it displayed an enhanced affinity for the agonist DAMGO similar to that of the high-affinity state of the wild type, which was not altered by GTPgammaS, while having unchanged affinity for the antagonist diprenorphine. The T6.34(279)K mutant displayed a higher intracellular receptor pool than the wild type. Naloxone inhibited the basal [35S]GTPgammaS binding of the T6.34(279)K mutant, demonstrating inverse agonist activity at this mutant receptor. In contrast, the T6.34(279)D substitution did not increase basal [35S]GTPgammaS binding, greatly reduced agonist-promoted [35S]GTPgammaS binding, and markedly decreased affinity for DAMGO. Thus, the T6.34(279)D mutant adopts conformations corresponding to inactive states of the receptor. The results were interpreted in the structural context of a model for the mu opioid receptor that incorporates the information from the crystal structure of rhodopsin. The interaction of T6.34(279) with R3.50(165) in the mu opioid receptor is considered to stabilize the inactive conformations. The T6.34(279)K substitution would then disrupt this interaction and support agonist-free activation, while T6.34(279)D mutation should strengthen this interaction which keeps the receptor in inactive states. T6.34(279) may, in addition, interact with the neighboring R6.35(280) to help constrain the receptor in inactive states, and T6.34(279)K and T6.34(279)D mutations would affect this interaction by disrupting or strengthening it, respectively. To the best of our knowledge, the results presented here represent the first structurally rationalized demonstration that mutations of this locus can lead to dramatically different properties of a GPCR.
Assuntos
Motivos de Aminoácidos/fisiologia , Receptores Opioides mu/fisiologia , Motivos de Aminoácidos/genética , Substituição de Aminoácidos , Animais , Ácido Aspártico/genética , Células CHO , Sequência Conservada , Cricetinae , Diprenorfina/metabolismo , Expressão Gênica/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Lisina/genética , Modelos Moleculares , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Toxina Pertussis , Ratos , Receptores Opioides mu/química , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Radioisótopos de Enxofre , Treonina/genética , Trítio , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Adenosine-to-inosine RNA editing events that have been demonstrated for 5HT (2C) receptors resulted in alterations of the amino acid sequence at positions 156, 158 and 160 in the intracellular loop 2 (IL2) region. The edited receptor isoforms were shown to have reduced basal activity, but similar maximum responses to agonist binding. To identify the molecular mechanism of these pharmacological effects of editing we explored the conformational properties of the edited IL2 in comparison with the wild type. The results from conformational studies of the IL2 isoforms, using biased Monte Carlo simulations with an implicit solvent model based on a screened Coulomb potential, show that the compared loops differ in their preferred spatial orientations as a result of differences in the conformational space that is accessible to them by energy criteria. For the IL2 of the unedited (5HT (2C-INI) ) receptor, the preference for structures oriented towards the 7TM bundle is larger than for the 5HT (2C-VGV) edited receptor. This difference in preferred orientation can affect the association of IL2 with other intracellular loop domains involved in G protein coupling and hence the coupling efficiency. The results illustrate the high sensitivity of the system to small changes in the interaction surface presented to other intracellular loops, and/or the G protein.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Edição de RNA , Receptores de Serotonina/química , Receptores de Serotonina/metabolismo , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Método de Monte Carlo , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/química , Estrutura Terciária de Proteína , Receptor 5-HT2C de Serotonina , Relação Estrutura-AtividadeRESUMO
Previously, we have identified three Zn(2+) binding residues in an endogenous Zn(2+) binding site in the human dopamine transporter (hDAT): (193)His in extracellular loop 2 (ECL 2), (375)His at the external end of transmembrane segment (TM) 7, and (396)Glu at the external end of TM 8. Here we have generated a series of artificial Zn(2+) binding sites in a domain situated around the external ends of TMs 7 and 8 by taking advantage of the well-defined structural constraints for binding of the zinc(II) ion. Initially, we found that the Zn(2+)-coordinating (193)His in ECL 2 could be substituted with a histidine inserted at the i - 4 position relative to (375)His in TM 7. In this mutant (H193K/M371H), Zn(2+) potently inhibited [(3)H]dopamine uptake with an IC(50) value of 7 microM as compared to a value of 300 microM for the control (H193K). These data are consistent with the presence of an alpha-helical configuration of TM 7. This inference was further corroborated by the observation that no increase in the apparent Zn(2+) affinity was observed following introduction of histidines at the i - 2, i - 3, and i - 5 positions. In contrast, introduction of histidines at positions i + 2, i + 3, and i + 4 all resulted in potent inhibition of [(3)H]dopamine uptake by Zn(2+) (IC(50) = 3-32 microM). These observations are inconsistent with continuation of the helix beyond position 375 and indicate an approximate boundary between the end of the helix and the succeeding loop. In summary, the data presented here provide new insight into the structure of a functionally important domain in the hDAT and illustrate how engineering of Zn(2+) binding sites can be a useful approach for probing both secondary and tertiary structure relationships in membrane proteins of unknown structure.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Engenharia de Proteínas/métodos , Zinco/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Ligação Competitiva/genética , Células COS , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Simulação por Computador , Proteínas da Membrana Plasmática de Transporte de Dopamina , Histidina/genética , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Zinco/metabolismoRESUMO
The binding site of the dopamine D2 receptor, like that of homologous G-protein-coupled receptors (GPCRs), is contained within a water-accessible crevice formed among its seven transmembrane segments (TMSs). Using the substituted-cysteine-accessibility method (SCAM), we are mapping the residues that contribute to the surface of this binding-site crevice. We have mutated to cysteine, one at a time, 21 consecutive residues in the fourth TMS (TM4). Eleven of these mutants reacted with charged sulfhydryl-specific reagents, and bound antagonist protected nine of these from reaction. For the mutants in which cysteine was substituted for residues in the cytoplasmic half of TM4, treatment with the reagents had no effect on binding, consistent with these residues being inaccessible and with the low-resolution structure of the homologous rhodopsin, in which TM3 and TM5 occlude the cytoplasmic half of TM4. Although hydrophobicity analysis positions the C-terminus of TM4 at 4.64, Pro-Pro and Pro-X-Pro motifs, which are known to disrupt alpha-helices, occur at position 4.59 in a number of homologous GPCRs. The SCAM data were consistent with a C-terminus at 4.58, but it is also possible that the alpha-helix extends one additional turn to 4.62 in the D2 receptor, which has a single Pro at 4.59. In homologous GPCRs, the high degree of sequence variation between 4.59 and 4.68 is more characteristic of a loop domain than a helical segment. This region is shown here to be very conserved within functionally related receptors, suggesting an important functional role for this putative nonhelical domain. This inference is supported by observed ligand-specific effects of mutations in this region and by the predicted spatial proximity of this segment to known ligand binding sites in other TMs.
Assuntos
Fragmentos de Peptídeos/química , Receptores de Dopamina D2/química , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sítios de Ligação/genética , Linhagem Celular , Membrana Celular/química , Membrana Celular/genética , Sequência Conservada , Cisteína/genética , Espaço Extracelular/química , Espaço Extracelular/genética , Humanos , Indicadores e Reagentes , Mesilatos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/genética , Prolina/química , Prolina/genética , Estrutura Secundária de Proteína/genética , Receptores de Dopamina D2/genética , Proteínas Recombinantes/química , Análise de Sequência de Proteína , Eletricidade Estática , Moldes GenéticosRESUMO
Proline residues are known to perturb the structure of helices by introducing a kink between the segments preceding and following the proline residue. The distortion of the helical structure results from the avoided steric clash between the ring of the proline at position (i) and the backbone carbonyl at position (i - 4), as well as the elimination of helix backbone H-bonds for the carbonyls at positions (i - 3) and (i - 4). Both the departure from the ideal helical pattern and the reduction in H-bond stabilization contribute to the observed flexibility of a proline-containing alpha-helix. The special local flexibility of the proline kink can confer an important role on the proline-containing helix in the conformational changes related to the function of the protein. As a useful tool in determining and evaluating the role of proline-induced flexibility and distortions in protein function, we present here a protocol to quantify the geometry of the distortion introduced in helices by prolines both as a time-averaged value and for individual 'snapshots' along a molecular dynamics simulation.