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PubMed1998

Dissection of de novo membrane insertion activities of internal transmembrane segments of ATP-binding-cassette transporters: toward understanding topological rules for membrane assembly of polytopic membrane proteins.

The membrane assembly of polytopic membrane proteins is a complicated process. Using Chinese hamster P-glycoprotein (Pgp) as a model protein, we investigated this process previously and found that Pgp expresses more than one topology. One of the variations occurs at the transmembrane (TM) domain including TM3 and TM4: TM4 inserts into membranes in an N(in)-C(out) rather than the predicted N(out)-C(in) orientation, and TM3 is in cytoplasm rather than the predicted N(in)-C(out) orientation in the membrane. It is possible that TM4 has a strong activity to initiate the N(in)-C(out) membrane insertion, leaving TM3 out of the membrane. Here, we tested this hypothesis by expressing TM3 and TM4 in isolated conditions. Our results show that TM3 of Pgp does not have de novo N(in)-C(out) membrane insertion activity whereas TM4 initiates the N(in)-C(out) membrane insertion regardless of the presence of TM3. In contrast, TM3 and TM4 of another polytopic membrane protein, cystic fibrosis transmembrane conductance regulator (CFTR), have a similar level of de novo Nin-Cout membrane insertion activity and TM4 of CFTR functions only as a stop-transfer sequence in the presence of TM3. Based on these findings, we propose that 1) the membrane insertion of TM3 and TM4 of Pgp does not follow the sequential model, which predicts that TM3 initiates N(in)-C(out) membrane insertion whereas TM4 stops the insertion event; and 2) "leaving one TM segment out of the membrane" may be an important folding mechanism for polytopic membrane proteins, and it is regulated by the N(in)-C(out) membrane insertion activities of the TM segments.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Development of a self-proliferating Leydig cell line: a hyper-sensitive E-screening model.

The mechanisms of estrogenic endocrine disruption on the male reproductive tract are poorly understood. In order to examine estrogenic properties of xenobiotic chemicals on male tissues, we have developed a mouse Leydig cell line (TM3-SF) that self-proliferates under serum-free conditions. This cell line was derived from ATCC's cell line, TM3. The development of TM3-SF was accomplished over a 4-month period by a progressive serum starvation of the original TM3 cells. The newly established cell line was maintained under serum-free conditions for 20 passages prior to testing. Sensitivity of the TM3-SF cells to estrogens was assayed by cell proliferation studies. A total of four compounds, diethylstilbestrol (DES), 17beta-estradiol, 17alpha-estradiol, and Bis-phenol A, were tested. Significant increases in cell proliferation occurred at various concentrations ranging from 1 pg/ml to 100 ng/ml for all four compounds. The order of potency observed was DES > Bis-phenol A > 17beta-estradiol and > 17alpha-estradiol. In addition, we investigated the mechanism for the self-proliferative properties of TM3-SF. The results of these trials indicate that either inhibin or activin is a primary growth factor for this cell line as a 50% inhibition of growth was noted when cell cultures were exposed to the anti-betaa subunit of inhibin/activin. Furthermore, the addition of the anti-betaa subunit of inhibin/activin blocked the DES-induced proliferation of TM3-SF. We conclude that the growth of TM3-SF cells is estrogen sensitive and that either inhibin or activin is involved in the self-regulation of growth.

Activins↗
PubMed2006

Structure and pH sensitivity of the transmembrane segment 3 of rhodopsin.

Activation of G protein-coupled receptors (GPCRs) originates in ligand-induced protein conformational changes that are transmitted to the cytosolic receptor surface. In the photoreceptor rhodopsin, and possibly other rhodopsin-like GPCRs, protonation of a carboxylic acid in the conserved E(D)RY motif at the cytosolic end of transmembrane helix 3 (TM3) is coupled to receptor activation. Here, we have investigated the structure of synthetic peptides derived from rhodopsin TM3. Polarized FTIR spectroscopy reveals a helical structure of a 31-mer TM3 peptide reconstituted into PC vesicles with a large tilt of 40-50 degrees of the helical axis relative to the membrane normal. Helical structure is also observed for the TM3 peptide in detergent micelles and depends on pH, especially in the C-terminal sequence. In addition, the fluorescence emission of the single tyrosine of the D(E)RY motif in the TM3 peptide exhibits a pronounced pH sensitivity that is abolished when Glu is replaced by Gln, demonstrating that protonation of the conserved Glu side chain affects the structure in the environment of the D(E)RY motif of TM3. The pH regulation of the C-terminal TM3 structure may be an intrinsic feature of the E(D)RY motif in other class I receptors, allowing the coupling of protonation and conformation of membrane-exposed residues in full-length GPCRs.

Hydrogen-Ion Concentration↗
PubMed2005

Structure and dynamics of the second and third transmembrane domains of human glycine receptor.

A 61-residue polypeptide resembling the second and third transmembrane domains (TM23) of the alpha-1 subunit of human glycine receptor and its truncated form, both with the wild-type loop linking the two TM domains (the "23" loop), were studied using high-resolution NMR. Well-defined domain structures can be identified for the TM2, 23 loop, and TM3 regions. Contrary to the popular model of a long and straight alpha-helical structure for the pore-lining TM2 domain for the Cys-loop receptor family, the last three residues of the TM2 domain and the first eight residues of the 23 loop (S16-S26) seem to be intrinsically nonhelical and highly flexible even in trifluoroethanol, a solvent known to promote and stabilize alpha-helical structures. The six remaining residues of the 23 loop and most of the TM3 domain exhibit helical structures with a kinked pi-helix (or a pi-turn) from W34 to C38 and a kink angle of 159 +/- 3 degrees . The tertiary fold of TM3 relative to TM2 is defined by several unambiguously identified long-range NOE cross-peaks within the loop region and between TM2 and TM3 domains. The 20 lowest-energy structures show a left-handed tilt of TM3 relative to TM2 with a tilting angle of 44 +/- 2 degrees between TM2 (V1-Q14) and TM3 (L39-E48) helix axes. This left-handed TM2-TM3 arrangement ensures a neatly packed right-handed quaternary structure of five subunits to form an ion-conducting pore. This is the first time that two TM domains of the glycine receptor linked by the important 23 loop have ever been analyzed at atomistic resolution. Many structural characteristics of the receptor can be inferred from the structural and dynamical features identified in this study.

Amides↗
PubMed1988

Cloning and characterization of a cDNA encoding transformation-sensitive tropomyosin isoform 3 from tumorigenic human fibroblasts.

We isolated a cDNA clone from the tumorigenic human fibroblast cell line HuT-14 that contains the entire protein coding region of tropomyosin isoform 3 (Tm3) and 781 base pairs of 5'- and 3'-untranslated sequences. Tm3, despite its apparent smaller molecular weight than Tm1 in two-dimensional gels, has the same peptide length as Tm1 (284 amino acids) and shares 83% homology with Tm1. Tm3 cDNA hybridized to an abundant mRNA of 1.3 kilobases in fetal muscle and cardiac muscle, suggesting that Tm3 is related to an alpha fast-tropomyosin. The first 188 amino acids of Tm3 are identical to those of rat or rabbit skeletal muscle alpha-tropomyosin, and the last 71 amino acids differ from those of rat smooth muscle alpha-tropomyosin by only 1 residue. Tm3 therefore appears to be encoded by the same gene that encodes the fast skeletal muscle alpha-tropomyosin and the smooth muscle alpha-tropomyosin via an alternative RNA-splicing mechanism. In contrast to Tm4 and Tm5, Tm3 has a small gene family, with, at best, only one pseudogene.

Amino Acid Sequence↗
PubMed1980

Calcium and pancreatic beta-cell function. IX. Demonstration of lanthanide-induced inhibition of insulin secretion independent of modifications in transmembrane Ca2+ fluxes.

beta-Cell-rich pancreatic islets were microdissected from noninbred ob/ob-mice and used to examine the mode of action of trivalent lanthanide ions on insulin secretion. La3+, Sm3+, and Tm3+ were equally effective inhibitors of basal and glucose-stimulated insulin release. As indicated by perifusion experiments with Tm3+, the inhibitory action was prompt, sustained, and readily reversible. Despite the similarities among the lanthanides in inhibiting insulin secretion, these cations differed considerably in their ability to impair transmembrane 45Ca fluxes. Using 10 different members of the lanthanide series, it was possible to demonstrate that their effectiveness to inhibit 45Ca uptake increased with ionic radius. La3+ markedly inhibited intracellular uptake and superficial binding of 45Ca at both 3 and 20 mM glucose. However, Tm3+ failed to affect intracellular 45Ca uptake and only reduced superficial binding of 45Ca at 3 mM glucose. In efflux experiments, Tm3+ did not affect basal or glucose-stimulated 45Ca washout from islets perifused with a medium containing 1.28 mM Ca2+. In a Ca2+-deficient medium, Tm3+ caused a slight transient increase, followed by reduction of 45Ca washout. However, when glucose was omitted, there was a prompt increase in the washout of radioactivity in the presence of Tm3+. Accordingly, the potent inhibitory action of Tm3+ on insulin secretion is not matched by changes in transmembrane Ca2+ fluxes. Since the lanthanides do not penetrate intracellularly, we propose the existence of cationic binding sites in the beta-cell plasma membrane with direct inhibitory effects on insulin secretion.

Animals↗
PubMed2006

Effects of human pregnancy and advancing gestation on respiratory discomfort during exercise.

This study examined the effects of human pregnancy and advancing gestation on the intensity of respiratory discomfort (dyspnea) during cycle exercise. Fourteen pregnant women (PG) performed a progressive cycle ergometer exercise test involving 20 W/min increases in work rate to symptom limitation and/or a heart rate of 170-175 beats/min at 19.7+/-1.2 weeks (ENTRY), 28.2+/-0.3 weeks (TM2) and 36.3+/-0.3 weeks (TM3) gestation. Eight, age-matched, sedentary non-pregnant women (CG) were also studied for comparison purposes. Measurements included dyspnea intensity (Borg scale), minute ventilation (VE), breathing pattern and other cardiorespiratory parameters. At peak exercise, neither pregnancy nor advancing gestation had an effect on dyspnea, VE, breathing pattern, oxygen uptake or work rate (p>0.05). VE was significantly greater (by 11 L/min at 100 W) in the PG at TM3 versus CG (p<0.05) at all submaximal work rates. VE also increased progressively from ENTRY to TM2 and TM3 during submaximal exercise. Dyspnea was not significantly different at any submaximal work rate in the PG at TM3 versus CG or with advancing gestation in the PG. In addition, dyspnea at a standardized exercise VE of 40 L/min was not different at TM3 versus ENTRY or in the PG at TM3 versus CG. Neither pregnancy nor advancing gestation were associated with increased respiratory discomfort during strenuous non-weight bearing cycle ergometer exercise, despite substantial increases in VE and progressive mechanical adaptations of the respiratory system to accommodate the increasing size of the gravid uterus.

Analysis of Variance↗
PubMed1999

Subunit mutations affect ethanol actions on GABA(A) receptors expressed in Xenopus oocytes.

1. Mutations of specific amino acids were introduced in transmembrane domains (TM) of GABA(A) receptor alpha2, beta1 and gamma2L subunits. The effects of these mutations on the action of ethanol were studied using the Xenopus oocyte expression system and two-electrode voltage-clamp recording techniques. 2. Mutant alpha2 subunits containing S270I (TM2) or A291W (TM3) made the receptor more sensitive to GABA, as compared to wild-type alpha2beta1gamma2L receptor. The mutation S265I (TM2) of beta1 and S280I (TM2) or S30IW (TM3) in gamma2L subunits did not alter apparent affinity of the receptor for GABA. M286W (TM3) in the beta1 subunit resulted in a receptor that was tonically open. 3. Using an EC5 concentration of GABA, the function of the wild-type receptor with alpha2beta1gamma2L subunits was potentiated by ethanol (50-200 mM). The mutations in TM2 or TM3 of the alpha2 subunit diminished the potentiation by ethanol. The action of ethanol was also eliminated with a mutation in the TM2 site of the beta1 subunit. Ethanol produced significant inhibition of GABA responses in receptors containing the combination of alpha2 and beta1 TM2 mutants with a wild-type gamma2L subunit. A small but significant reduction in the potentiation by ethanol was observed with gamma2L TM2 and/or TM3 mutants. 4. From these results, we suggest that in heteromeric GABA(A) receptors composed of the alpha, beta and gamma subunits, ethanol may bind in a cavity formed by TM2 and TM3, and that binding to the alpha or beta subunit may be more critical than the gamma subunit.

Amino Acid Sequence↗
PubMed2006

Effects of amino acid substitutions in transmembrane domains of the NR1 subunit on the ethanol inhibition of recombinant N-methyl-D-aspartate receptors.

BACKGROUND: The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is involved in a variety of processes that regulate neuronal plasticity and is an important target for the acute and chronic effects of ethanol. However, the specific sites where ethanol interacts with the receptor protein have yet to be fully elucidated. We previously demonstrated that a phenylalanine to alanine mutation in the third transmembrane domain (TM3) of the NR1 subunit decreased the ethanol inhibition of NMDA receptors expressed in HEK293 cells. In this study, we characterized the ethanol inhibition of NMDA receptors containing additional mutations within the TM3 and TM4 domains of the NR1 subunit. METHODS: Site-directed mutagenesis was used to alter specific amino acid residues in the TM3 and TM4 domains of the NR1 subunit. Mutant NR1 subunits were coexpressed with the NR2A subunit in HEK293 cells and examined for alterations in ethanol sensitivity using whole-cell voltage-clamp electrophysiology. RESULTS: Replacing phenylalanine at TM3 position 639 in the NR1 subunit (F639) with 9 different amino acids produced functional receptors when coexpressed with the NR2A subunit. All mutants showed a concentration-dependent inhibition by ethanol (10-100 mM), with the alanine and serine mutants being significantly less sensitive to ethanol. Amino acid substitutions at the F639 site also produced variable changes in the concentration-response relationship to glycine. However, no significant correlation between glycine EC(50) values and the magnitude of ethanol inhibition was observed. Alanine mutations at TM4 positions 813 (M813A) and 819 (L819A), but not at 817 (F817A), of the NR1 subunit enhanced ethanol inhibition. Substitution of tryptophan for TM4 residues in the NR1 subunit (positions 820-822) that are homologous to a site in the NR2A subunit shown to reduce ethanol inhibition (A825W) had no effect on ethanol sensitivity. However, these NR1 TM4 tryptophan mutants restored the ethanol inhibition of the NR1 TM3 F639A mutant to wild-type levels in a stepwise fashion. CONCLUSIONS: These results indicate that the ethanol sensitivity of NMDA receptors may be regulated by discrete sites within the TM3 and TM4 domains of the NR1 subunit.

Amino Acid Substitution↗
PubMed2004

Mutational analysis of transmembrane regions 3 and 4 of SecY, a central component of protein translocase.

The SecYEG heterotrimeric membrane protein complex functions as a channel for protein translocation across the Escherichia coli cytoplasmic membrane. SecY is the central subunit of the SecYEG complex and contains 10 transmembrane segments (TM1 to TM10). Previous mutation studies suggested that TM3 and TM4 are particularly important for SecY function. To further characterize TM3 and TM4, we introduced a series of cysteine-scanning mutations into these segments. With one exception (an unstable product), all the mutant proteins complemented the cold-sensitive growth defect of the secY39 mutant. A combination of this secY mutation and the secG deletion resulted in synthetic lethality, and the TM3 and TM4 SecY cysteine substitution mutations were examined for their ability to complement this lethality. Although they were all positive for complementation, some of the complemented cells exhibited significant retardation of protein export. The substitution-sensitive residues in TM3 can be aligned to one side of the alpha-helix, and those in TM4 revealed a tendency for residues closer to the cytosolic side of the membrane to be more severely affected. Disulfide cross-linking experiments identified a specific contact point for TM3 and SecG TM2 as well as for TM4 and SecG TM1. Thus, although TM3 and TM4 do not contain any single residue that is absolutely required, they include functionally important helix surfaces and specific contact points with SecG. These results are discussed in light of the structural information available for the SecY complex.

Adenosine Triphosphatases↗
PubMed1984

[Echocardiography study of hemodynamic changes in pregnancy].

The purpose of this study is to monitor the haemodynamic changes that occur in pregnancy using ultrasound. Although all previous studies do demonstrate an increase in frequency and the volume of the cardiac output, the results seem to be different as far as variations in volume during systole occur and the mechanisms by which they occur. These differences can be explained by problems associated with the different methods used. Our study was carried out on ten pregnant women who were examined in each trimester (TM1, TM2, TM3) and post-partum (TM4). The results confirm that cardiac frequency rises (TM3 92 +/- 5 VS TM4 73 +/- 3 cpm p less than 0,01) and cardiac output does (TM3 7,2 +/- 0,6 VS TM4 4,8 +/- 0,3 Umm p less than 0,01). The volume in systolic ejection (VES) rises throughout pregnancy when the patient is lying on her left side (TM3 81 +/- 4 VS TM4 69 +/- 2 ml p less than 0,01). The rise in VES could be put down to an increase to the veinous return which itself is reflected by the left auricle (TM3 33,7 +/- 1,1 mm VS TM4 29,9 +/- 0,7 mm p less than 0,05) and in the left ventricle (TM3 48,7 +/- 0,6 VS TM4 46,3 +/- 0,3 mm p less than 0,01) and the sudden drop in the total peripheral resistance (TM1 950 +/- 50 VS TM4 1 335 +/- 95 dynes/sec. X cm-5 p less than 0,01). It does not seem as though there is any change in myocardial contractility.

Echocardiography↗
PubMed1994

Transmembrane orientation and topogenesis of the third and fourth membrane-spanning regions of human P-glycoprotein (MDR1).

Understanding how the multidrug resistance phenotype is manifest in human cancer cells will require insight into the mechanism of assembly, transmembrane topology, and intracellular trafficking of human P-glycoprotein (MDR1). Previously, we showed that MDR1 amino terminus biogenesis occurred through an unexpected interaction between novel topogenic sequence subtypes and that transmembrane topology of corresponding amino and carboxy halves was not equivalent. We now investigate topology and topogenic activities of the third and fourth transmembrane regions (TM3 and TM4) of human MDR1 using protease protection of defined reporter epitopes expressed in Xenopus laevis oocytes. As was previously observed for TM1 and TM2, determinants in TM3 and TM4 exhibited cooperativity in directing proper assembly and transmembrane orientation. The signal sequence encompassing TM3 required residues from TM4 to reinitiate translocation of the MDR1 chain into the endoplasmic reticulum (ER) lumen. Remaining residues from TM4 terminated translocation and established a polytopic transmembrane topology in which TM3 and TM4 both spanned the membrane in the orientation predicted by hydropathy-based models. Remarkably, when translocating sequentially into the ER lumen, neither TM4 alone nor TM4 together with TM3 efficiently terminated translocation. Thus, MDR1 biogenesis required both the presence of these sequences and their proper orientation with respect to the ER translocation apparatus. This conclusion was supported by experiments in which TM3 and TM4 topology was reproduced in a defined chimeric protein which mimicked native MDR1 presentation. These additional variations on simple themes of protein topogenesis utilized by MDR1 demonstrate that events of complex protein biogenesis may be dissected and studied using protein chimeras with defined translocation properties.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Membrane topogenesis of the three amino-terminal transmembrane segments of glucose-6-phosphatase on endoplasmic reticulum.

We investigated the membrane topogenesis of glucose-6-phosphatase (G6Pase), a multispanning membrane protein, on the endoplasmic reticulum. In COS-7 cells, the first transmembrane segment (TM1) with weak hydrophobicity is inserted into the membrane in the N-terminus-out/C-terminus-cytoplasm orientation. The following TM2 is inserted depending on TM3. TM3 has the same orientation as TM1. In contrast to data from living cells, the full-length molecule and N-terminal fusion constructs were not inserted into the membrane in a cell-free system. Addition of a signal recognition particle did not improve G6Pase insertion. When the 37-residue N-terminal segment was deleted, however, TM2 and TM3 were correctly inserted. We concluded that the three N-terminal TM segments are inserted into the membrane dependent on the two signal-anchor sequences of TM1 and TM3. TM1 is likely to be an unconventional signal sequence that barely functions in vitro. The 37-residue N-terminal segment inhibits the signal function of the following TM3 in cell-free systems.

Amino Acid Sequence↗
PubMed1995

Expression of the mouse mammary tumor virus long terminal repeat open reading frame promotes tumorigenic potential of hyperplastic mouse mammary epithelial cells.

The retrovirus mouse mammary tumor virus (MMTV) 3' long terminal repeat (LTR) contains an open reading frame (ORF) for a 36-kDa protein and encodes a superantigen activity [pORF(sag)]. We have tested the potential oncogenic activity of pORF(sag) in two immortalized mouse mammary epithelial cells. We subcloned MMTV LTR ORF DNA into the pRc/CMV mammalian expression vector in order to place LTR ORF transcription under the control of the constitutive CMV promoter. Mouse mammary epithelial cell lines TM3 and FSK7e4 were transfected and G418-resistant cell clones were isolated. Reverse transcription-polymerase chain reaction and Northern blot analyses revealed modest overexpression of LTR RNA in several transfected cell clones of each line. Individual cell clones were transplanted into cleared mammary gland fat pads of syngeneic BALB/c mice. The parental cell lines and FSK7e4-derived clones did not form tumors, whereas ORF-transfected clones derived from the TM3 cells formed tumors within 8 weeks in 100% of transplanted fat pads in multiple experiments. The tumor cells expressed exogenous LTR ORF RNA and were proven to be derivatives of TM3 cells based on a marker p53 mutation. Immunohistochemistry using a polyclonal antiserum raised against pORF(sag) expressed in insect cells revealed a cytoplasmic reaction in TM3-CMV-LTR tumor cells; a much weaker cytoplasmic reaction was detected in the transfected tissue culture cells. These observations suggest that MMTV pORF(sag) may act as an oncogene in certain mouse mammary epithelial cells and raise the possibility that pORF(sag) may have a role in mammary tumorigenesis. As the parental FSK7 cell line has produced only ductal outgrowths upon transplantation in vivo and the TM3 cell line produces a nontumorigenic hyperplasia, the results suggest further that pORF(sag) may influence the latter stages of mammary tumorigenesis, namely, the preneoplastic to neoplastic transformation.

Animals↗
PubMed1990

Differential regulation and expression of major histocompatibility complex (MHC) and Ly-6 gene products on mouse testicular Leydig and Sertoli cell lines.

The expression and regulation of Class I and Class II major histocompatibility complex (MHC) and Ly-6 antigens were examined in BALB/c testicular cells. Studies were performed utilizing differentiated murine Leydig (TM3) and Sertoli (TM4) cell lines. Neither Class I (Dd) nor Class II (IA/Ed) MHC antigens were detectable on untreated TM3 cells. However, concanavalin-A activated spleen cell supernatant (Con-A sup) or interferon-gamma (IFN-gamma) treatment resulted in the marked induction of both Class I and Class II MHC antigens on virtually all of the Leydig cells. MHC Class II mRNA, which was not detected in resting cells, was clearly induced following IFN-gamma incubation. Sertoli cells were found to constitutively express low levels of Class I (Dd) but not Class II (IA/Ed) antigens. However, in contrast to the enhanced MHC expression in TM3 cells, Con-A sup or IFN-gamma treatment of TM4 cells resulted in marked augmentation of Class I, but not Class II, MHC antigens. Northern blot analysis failed to detect Class II mRNA in either the resting or IFN-gamma treated TM4 populations. Neither ethanol nor tumor necrosis factor (TNF) alone, or together with IFN-gamma head significant effects on MHC expression by TM3 and TM4 cells. Ly-6 antigens, predominantly expressed on hematopoietic cells, were found to be present on both TM3 and TM4 cells. Expression of this non-MHC encoded product was also shown to be markedly enhanced by IFN-gamma treatment on both testicular cell lines. In total, these findings demonstrated that cytokines can differentially affect discrete cell populations arising from a particular tissue with respect to the un-regulation of MHC and non-MHC gene products. These findings are discussed in the context of autoimmune responses directed against this tissue.

Animals↗
PubMed1999

Molecular modeling of the dopamine D2 and serotonin 5-HT1A receptor binding modes of the enantiomers of 5-OMe-BPAT.

Molecular modeling studies were undertaken in order to elucidate the possible dopamine D2 and serotonin 5-HT1A receptor binding modes of the enantiomers of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1). For this purpose, a combination of indirect molecular modeling and direct construction of the seven transmembrane (7TM) domains of the receptors was employed in a stepwise, objective manner. Pharmacophore models and corresponding receptor maps were identified by superimposing selected sets of receptor agonists in their presumed pharmacologically active conformations, while taking the conformational freedom of the ligands into account. The 7TM models were then constructed around the agonist pharmacophore models, by adding the TM domains one-by-one. Initially, the relative positions of TM3, TM4, and TM5 were determined using the three-dimensional structure of bacteriorhodopsin, but subsequently the orientations of all TM domains were adjusted in order to mimic the topology of the TM domains of rhodopsin. The presumed dopamine D2 receptor binding conformations of (S)- and (R)-1 were determined by using the semirigid dopamine D2 receptor antagonist N-benzylpiquindone as a template for superposition. Similarly, the selective serotonin 5-HT1A receptor agonist flesinoxan was employed for identifying the serotonin 5-HT1A receptor binding conformations of the enantiomers of 1. After docking of the presumed pharmacologically active conformations in the 7TM models and subsequent optimization of the binding sites, specific interactions between the ligands and the surrounding amino acid residues, consistent with the structure-activity relationships, were observed. Thus, both enantiomers of 1 bound to the dopamine D2 receptor model in a similar fashion: a reinforced electrostatic interaction was present between the protonated nitrogen atoms and Asp114 in TM3; their carbonyl groups accepted a H-bond from Ser121 in TM3; their amide NH groups acted as H-bond donor to Tyr416 in TM7; and their benzamide phenyl rings were involved in a hydrophobic edge-to-face interaction with Trp386 in TM6. Differences were observed in the orientations of the 2-aminotetralin moieties, which occupied the agonist binding site. Whereas the (S)-enantiomer could form a H-bond between its 5-methoxy substituent and Ser193 in TM5, the (R)-enantiomer could not, which may account for the differences in their intrinsic efficacies at the dopamine D2 receptor. In the serotonin 5-HT1A receptor model, the benzamide phenyl rings of both enantiomers were involved in hydrophobic face-to-face interactions with Phe112 in TM3, while their protonated nitrogen atoms formed a reinforced electrostatic interaction with Asp116 in TM3. Consistent with the structure-affinity relationships of 1, the amide moieties were not involved in specific interactions. Both enantiomers of 1 could form a hydrogen bond between their 5-methoxy substituent and Thr200 in TM5, which may account for their full serotonin 5-HT1A receptor agonist properties.

Amino Acid Sequence↗
PubMed2000

Molecular determinants of ligand binding to the human melanocortin-4 receptor.

To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.

Agouti-Related Protein↗
PubMed2003

High affinity agonistic metal ion binding sites within the melanocortin 4 receptor illustrate conformational change of transmembrane region 3.

We created a molecular model of the human melanocortin 4 receptor (MC4R) and introduced a series of His residues into the receptor protein to form metal ion binding sites. We were able to insert micromolar affinity binding sites for zinc between transmembrane region (TM) 2 and TM3 where the metal ion alone was able to activate this peptide binding G-protein-coupled receptor. The exact conformation of the metal ion interactions allowed us to predict the orientation of the helices, and remodeling of the receptor protein indicated that Glu100 and Ile104 in TM2 and Asp122 and Ile125 in TM3 are directed toward a putative area of activation of the receptor. The molecular model suggests that a rotation of TM3 may be important for activation of the MC4R. Previous models of G-protein-coupled receptors have suggested that unlocking of a stabilizing interaction between the DRY motif, in the cytosolic part of TM3, and TM6 is important for the activation process. We suggest that this unlocking process may be facilitated through creation of a new interaction between TM3 and TM2 in the MC4R.

Amino Acid Motifs↗