Human oxytocin receptors in cholesterol-rich vs. cholesterol-poor microdomains of the plasma membrane.

We analyzed the properties of a G protein-coupled receptor localized in cholesterol-poor vs. cholesterol-rich microdomains of the plasma membrane. For this purpose, the human oxytocin receptor, which is very sensitive against alterations of the membrane cholesterol level, was stably expressed in HEK293 cells. To calculate the total number of receptors independent of ligand binding studies, the oxytocin receptor was tagged with an enhanced green fluorescent protein (EGFP) which did not change the functional properties of the receptor. Only 1% of the oxytocin receptors were present in cholesterol-rich detergent-insoluble domains. In contrast, employing a detergent-free fractionation scheme that preserves the functional activity of the receptor, we detected 10-15% of the receptors in cholesterol-rich low-density membranes and therein the high-affinity state receptors were twofold enriched. In cholesterol-poor vs. cholesterol-rich domains, high-affinity oxytocin receptors behaved similar with respect to their agonist binding kinetics and GTP sensitivity. However, high-affinity oxytocin receptors localized in cholesterol-rich low-density membranes showed a markedly enhanced (t (1/2) approximately threefold) stability at 37 degrees C as compared with the oxytocin receptors localized in the cholesterol-poor high-density membranes. Addition of cholesterol to the high-density membranes fully protected the oxytocin receptors against loss of function. The importance of cholesterol to stabilize the oxytocin receptor was supported in experiments with solubilized receptors. Cholesterol markedly delayed the inactivation of oxytocin receptors solubilized with Chapso. In conclusion, the data of this report suggest that functional properties of heptahelical receptor proteins could differ in dependence of their localization in different membrane microdomains.     

Vasopressin and oxytocin receptors on plasma membranes from rat mammary gland. Demonstration of vasopressin receptors by stimulation of inositol phosphate formation, and oxytocin receptors by binding of a specific 125I-labeled oxytocin antagonist, d(CH2)5(1)[Tyr(Me)2, Thr4,Tyr-NH2(9)]OVT

The addition of oxytocin to minces of rat mammary gland preincubated with (3H)myo-inositol stimulated the formation of inositol phosphate (IP) in both lactating and regressed glands. Stimulation was about 4 times greater in regressed tissue, consistent with an oxytocin effect on myoepithelial cells, which are enriched relative to epithelial cells during regression. The stimulation of IP formation was agonist specific, as shown with several oxytocin analogs. Arginine vasopressin (AVP), however, was more than twice as potent as oxytocin in stimulating IP formation in regressed tissue. Both V1- and V2-selective AVP receptor antagonists inhibited the stimulation of IP formation by oxytocin. The V1-selective antagonist was about 10 times more inhibitory than the V2-selective antagonist. [3H]AVP was bound to plasma membranes from the mammary gland of the lactating rat with an apparent Kd of about 0.7 nM and Bmax of 54.6 fmol/mg protein. These values were comparable with those found for AVP receptors of kidney plasma membranes. Our results suggest that the stimulation of IP formation in rat mammary gland by oxytocin occurs through occupancy of AVP, and not oxytocin, receptor sites. A second aspect of these studies was to determine if a recently developed iodinated antagonist of oxytocin-induced uterine contractions could be used as a specific probe for oxytocin receptors in the rat mammary gland. Under steady state conditions, [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT was bound to a single class of independent binding sites in mammary gland plasma membrane from lactating rats with an apparent Kd of 65 pM and Bmax of 225 fmol/mg protein. Noniodinated antagonist had an affinity about 150 times less than the monoiodinated form. The affinity of binding sites for AVP was 10 times greater than the noniodinated antagonist and 2.4 times greater than oxytocin. In view of the presence of AVP receptors in mammary tissue, these findings suggested that the iodinated antagonist binds to AVP receptors. However, comparison of the binding of iodinated antagonist to plasma membranes from the lactating mammary gland with kidney medulla and liver, target sites for AVP, showed that binding was specific for the mammary gland and hence oxytocin receptors. The concentration of oxytocin receptors in mammary gland, as determined by [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT binding, was 4 times greater than the concentration of high-affinity AVP receptors, as determined by [3H]AVP binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct identification of human oxytocin receptor-binding domains using a photoactivatable cyclic peptide antagonist: comparison with the human V1a vasopressin receptor

Understanding of the molecular determinants responsible for antagonist binding to the oxytocin receptor should provide important insights that facilitate rational design of potential therapeutic agents for the treatment of preterm labor. To study ligand/receptor interactions, we used a novel photosensitive radioiodinated antagonist of the human oxytocin receptor, d(CH(2))(5) [Tyr(Me)(2),Thr(4),Orn(8),Phe(3(125)I,4N(3))-NH(2)9]vasotocin. This ligand had an equivalent high affinity for human oxytocin and V(1a) vasopressin receptors expressed in Chinese hamster ovary cells. Taking advantage of this dual specificity, we conducted photoaffinity labeling experiments on both receptors. Photolabeled oxytocin and V(1a) receptors appeared as a unique protein band at 70-75 kDa and two labeled protein bands at 85-90 and 46 kDa, respectively. To identify contact sites between the antagonist and the receptors, the labeled 70-75- and the 46-kDa proteins were cleaved with CNBr and digested with Lys-C and Arg-C endoproteinases. The fragmentation patterns allowed the identification of a covalently labeled region in the oxytocin receptor transmembrane domain III consisting of the residues Leu(114)-Val(115)-Lys(116). Analysis of contact sites in the V(1a) receptor led to the identification of the homologous region consisting of the residues Val(126)-Val(127)-Lys(128). Binding domains were confirmed by mutation of several CNBr cleavage sites in the oxytocin receptor and of one Lys-C cleavage site in the V(1a) receptor. The results are in agreement with previous experimental data and three-dimensional models of agonist and antagonist binding to members of the oxytocin/vasopressin receptor family.     

Organization of fusicoccin receptor in higher plant plasma membrane: relationship between affinity and molecular mass

Higher plant plasma membranes carry receptors of different affinity for the phytotoxin fusicoccin. Reception of fusicoccin involves proteins belonging to the highly conserved 14-3-3 family, but the complete structure of the fusicoccin receptor (FCR) is unknown. Using radiation inactivation analysis, we estimated the molecular masses of low-affinity and high-affinity FCR at 63 +/- 7 and 130 +/- 15 kD, respectively. The dose dependences of receptor inactivation indicate that microsomal specimens contain "silent" FCRs of 420 +/- 90 kD in amounts commensurate with that of the active FCRs. Both low- and high-affinity FCRs are inactivated by hydrolytic enzymes from the outer surface of the plasma membrane, and impairment of protoplast integrity causes an irreversible transition of the low-affinity binding site into the high-affinity one. A scheme is proposed for the organization of different types of FCR in the plasma membrane, implying that the membrane affinity for fusicoccin reflects the interaction between proteins in the FCR complex.     

Solubilization and reconstitution of dopamine D1 receptor from bovine striatal membranes: Effects of agonist and antagonist pretreatment

The bovine striatal dopamine D₁ receptor was solubilized with a combination of sodium cholate and NaCl in the presence of phospholipids, following treatment of membranes with a dopaminergic agonist (SKF-82526-J) or antagonist (SCH-23390). The solubilized receptors were subsequently reconstituted into lipid vesicles by gel-filtration. A comparison of ligand-binding properties shows that the solubilized and reconstituted receptors bound [³H]SCH-23390 to a homogeneous site in a saturable, stereospecific and reversible manner with a K_d of 0.95 and 1.1 nM and a B_max of 918 and 885 fmol/mg protein respectively for agonist- and antagonist-pretreated preparations. These values are very similar to those obtained for membrane-bound receptors. The competition of antagonists for [³H]SCH-23390 binding exhibited a clear D₁ dopaminergic order in the reconstituted preparation obtained from either agonist or antagonist-pretreated membranes, except that (+)butaclamol was about four-fold more potent thancis-flupentixol in displacing [³H]SCH-23390 binding in preparation obtained from agonist-pretreated membranes compared to antagonist-pretreated membranes. The agonist/[³H]SCH-23390 competition studies revealed the presence of a highaffinity component of agonist binding in both the reconstituted receptor preparations. The number of high-affinity agonist binding sites, however, is 40–80% higher in reconstituted preparation obtained from antagonist-treated membrane compared to that obrained from the agonist-treated membrane. In both the preparations, 100 M guanylylimidodiphosphate (Gpp(NH)p) completely abolished the high-affinity component of agonist binding compared to partial abolition in the native membranes, indicating a close association of a G-protein with the solubilized receptors. Whether the receptor was solubilized following agonist or antagonist preincubation of the membranes, the receptor-detergent complex eluted from a steric-exclusion HPLC column with an apparent molecular size of 360,000. Preincubation of the solubilized preparations with Gpp(NH)p had virtually no effect on the elution profile suggesting a lack of guanine nucleotide-dependent dissociation of G-protein receptor complex.     

Oxytocin receptors and contractile response of the myometrium after long term infusion of prostaglandin F2 alpha, indomethacin, oxytocin and an oxytocin antagonist in rats

Binding of [3H]oxytocin to isolated myometrial plasma membranes was not affected by the presence of prostaglandin (PG)F2 alpha or E2 in the incubation medium. Long-term treatment with PGF2 alpha or indomethacin had no effect on oxytocin receptor concentrations and dissociation constants of myometrial plasma membranes nor on maximal contractility or KM values of isolated uterine strips exposed to oxytocin. Infusion of oxytocin for 5 days in non-pregnant rats resulted in a decrease in oxytocin receptor concentrations in myometrial plasma membranes whereas the binding affinity to oxytocin was unaffected. Isolated uterine strips from similarly treated rats showed a reduced maximal contractile response to oxytocin and an elevated KM value, possibly indicating an influence of oxytocin on the coupling between receptor occupancy and contractility. Treatment for 5 days with desamino1-[D-Tyr(O-ethyl)2-Thr4-Orn8] oxytocin (an oxytocin antagonist) increased the concentration of myometrial oxytocin receptors. In addition KD values of these receptors were elevated. The present results indicate that prolonged exposure to oxytocin leads to a down-regulation of the myometrial receptor concentration, which is not caused by ligand-receptor interaction in itself. The concerted effect of oxytocin and prostaglandins on myometrial contraction does not appear to involve modulation of the oxytocin receptor by prostaglandins.     

Partial functional reconstitution of the cardiac muscarinic cholinergic receptor

Digitonin-solubilized cardiac muscarinic receptors were reconstituted by dialysis into human erythrocyte acceptor membranes which lack high-affinity muscarinic receptors. The number of receptors reconstituted was proportional to the quantity of soluble receptors added to the reconstitution system. Specific [3H](-)-quinuclidinyl benzilate binding to the reconstituted receptor was found to be saturable with a Kd (dissociation constant) equal to 48 +/- 4 pM and a Bmax (maximal density of binding sites) equal to 50 +/- 5 fmol/mg of protein. Competitive binding studies indicated that the reconstituted receptors showed stereoselectivity and drug specificity consistent with a high-affinity muscarinic receptor. Agonist binding to the reconstituted receptor was decreased by the addition of guanyl-5'-yl imidodiphosphate. Sixty per cent of the reconstituted receptors were found to be integral membrane proteins. The molecular weight of the reconstituted receptor as determined by sodium dodecyl sulfate-gel electrophoresis was 76,000 +/- 2,000 and was identical to the molecular weight of the muscarinic receptor in the original cardiac membranes. The data indicate that a partially functional, intact muscarinic receptor was reconstituted into human erythrocyte acceptor membranes and that membrane constituents may be required to stabilize the receptor in a high-affinity state for antagonists.     

Nongenomic inhibition of oxytocin binding by progesterone in the ovine uterus

Progesterone (P4) has been reported to inhibit oxytocin (OT) binding to its receptor in isolated murine endometrial membranes. The purpose of the present research was to 1). examine the in vivo and in vitro effect of P4 on the binding of OT to its receptor in the ovine endometrium and 2). determine whether the endometrial plasma membranes have high-affinity binding sites for P4. Ovariectomized ewes were pretreated with a sequence of estradiol-17beta (2 days) and P4 (5 days) before being treated with estradiol-17beta plus either vehicle (corn oil), P4, or P4 + mifepristone (RU 486) for 3 consecutive days. Treatment of ewes with 10 mg P4/day for 3 days suppressed binding of OT (P < 0.01) compared with that of controls, whereas concomitant treatment with the progestin antagonist RU 486 (10 mg/day) blocked the effect of P4. Similarly, incubation of endometrial plasma membranes with P4 (5 ng/ml) inhibited binding of OT (P < 0.05), whereas this effect of P4 was blocked by the presence of RU 486 (10 ng/ml). By radioreceptor assay, the endometrial plasma membranes were found to contain a high-affinity binding site for P4 and the progestin agonist promegestone (Kd 1.2 x 10-9 and 1.74 x 10-10M, respectively). Incubation of endometrial plasma membranes with P4 (5 ng/ml) significantly increased the concentration of progestin binding sites. Binding of labeled promegestone (R 5020) was competitively inhibited by excess unlabeled R 5020, P4, RU 486, and OT but not by estradiol-17beta, cortisol, testosterone, and arginine vasopressin. These data suggest a direct suppressive action of P4 on the binding of OT to OT receptors in the ovine endometrial plasma membrane.     

Identification of a myometrial oxytocin-receptor protein

The specific binding of [3H]oxytoxin to uterine membrane preparations derived from different species at late pregnancy was examined. The highest receptor density (bmax value) was found in membranes derived from the myometria of guinea pigs between day 60 post-conception (bmax = 3.6 +/- 0.1 pmol/mg) and day 65 (bmax = 4.4 +/- 0.1 pmol/mg). The similarity of Kd values for oxytocin binding (Kd = 2.6 +/- 0.2 nM) and for vasopressin binding (Kd = 2.1 +/- 0.4 nM) to the same membranes derived from a guinea pig myometrium indicate a homogeneous population of high-affinity binding sites which do not discriminate between these two hormones. Competitive binding experiments with specific oxytocin agonists containing either sarcosine or N-methylalanine in the place of Pro7 demonstrated that these myometrial receptors have the pharmacological properties of oxytocin receptors. The analogue of 1-deamino-[8-lysine]vasopressin containing a photoreactive azidophenylamidino group at the sidechain of Lys8 retained roughly the same receptor affinity as oxytocin. In photoaffinity labelling experiments with the tritium-labelled analogue a membrane protein from guinea pig myometrium with an apparent relative molecular mass Mr of 78,000 +/- 5000 (n = 13) was preferentially labelled. The labelling of this protein was completely suppressed by a 100-fold molar excess of either oxytocin, or [Sar7]oxytocin or [Thr4, Sar7]oxytocin, but not by other peptide hormones. These results provide evidence that the labelled 78,000-Mr protein is a myometrial oxytocin-receptor protein.     

Agonist-specific, high-affinity binding epitopes are contributed by an arginine in the N-terminus of the human oxytocin receptor

The effects of the peptide hormone oxytocin (OT) are mediated by the oxytocin receptor, which is a member of the G-protein-coupled receptor family. Defining differences between the binding of agonists and antagonists to the OTR, at the molecular level, is of fundamental importance to understanding OTR activation and to rational drug design. Previous reports have indicated that the N-terminus of the OTR is required for OT binding. The aim of this study was to identify which individual residues within the N-terminal domain of the human OTR provided these OT binding epitopes. A series of truncated OTRs and mutant receptor constructs with systematic alanine substitution were characterized with respect to their pharmacological profile and intracellular signaling capability. Although a number of residues within the OTR will be required for optimal OT-OTR interaction, our data establish that Arg(34) within the N-terminal domain contributes to high-affinity OT binding. Removal of Arg(34) by truncation or substitution resulted in a 2000-fold decrease in OT affinity. In addition, we show that the arginyl at this locus is required for high-affinity binding of agonists in general. However, the importance of Arg(34) is restricted to agonist interaction with the OTR, as it was not required for binding peptide antagonist or non-peptide antagonist. It is noteworthy that the corresponding Arg in the related rat V(1a) vasopressin receptor is also required for high-affinity agonist binding. This study defines, at the molecular level, the role of the N-terminus of the OTR in high-affinity agonist binding and identifies a key residue for this function.     

Binding of chemotactic peptide to the outer surface and to whole human spermatozoa with different affinity states

Binding of N-formyl-methionyl-L-leucyl-[³H]phenylalanine (fML[³H]Ph) to human ejaculated spermatozoa and to its isolated plasma membrane was studied. Our data confirm the presence of specific receptors for f-MLPh in the human spermatozoa and suggest that whole spermatozoa receptors exist in two affinity states, one high-affinity, low-capacity specific receptor (K_d = 12.3 ± 0.5 nM, n = 22,285 ± 65,008 binding sites per sperm cell) and a second one (K_d = 700 ± 47 nM) that is not saturable, indicating a low-affinity, high-capacity nonspecific site. In contrast, sperm membrane showed only one class of binding site (K_d = 6.4 ± 0.12 nM), which was statistically different from that of the high-affinity binding site of intact spermatozoa. To explain this difference we discuss the possibility that first, the two binding affinities represent two interconvertible states of a single receptor population, which, depending on the metabolic activity of spermatozoa, may change its physicochemical properties; or second, they reflect two different processes, binding and/or transport into the spermatozoa.     

Experimental and theoretical characterization of the high-affinity cation-binding site of the purple membrane.

Binding of Mn2+ or Mg2+ to the high-affinity site of the purple membrane from Halobacterium salinarium has been studied by superconducting quantum interference device magnetometry or by ab initio quantum mechanical calculations, respectively. The binding of Mn2+ cation, in a low-spin state, to the high-affinity site occurs through a major octahedral local symmetry character with a minor rhombic distortion and a coordination number of six. A molecular model of this binding site in the Schiff base vicinity is proposed. In this model, a Mg2+ cation interacts with one oxygen atom of the side chain of Asp85, with both oxygen atoms of Asp212 and with three water molecules. One of these water molecules is hydrogen bonded to both the nitrogen of the protonated Schiff base and the Asp85 oxygen. It could serve as a shuttle for the Schiff base proton to move to Asp85 in the L-M transition.     

Sodium Nitroprusside Induces Internalization of Muscarinic Receptors Stably Expressed in Chinese Hamster Ovary Cell Lines

Abstract: We have characterized the internalization of muscarinic acetylcholine receptors induced by the nitric oxide (NO)-generating compound sodium nitroprusside. When Chinese hamster ovary cells, stably transfected with the human m4 muscarinic receptor subtype, were incubated for 1 h in the presence of 700 µ M sodium nitroprusside, the number of receptors measured in intact cells with the hydrophilic ligand N -[³H]methylscopolamine was reduced by 30%. The effect was dose dependent, beginning with a concentration of sodium nitroprusside as low as 45 µ M . Removal of sodium nitroprusside from the incubation medium did not result in a recovery of the binding sites. The phenomenon was temperature dependent and was blocked by the muscarinic antagonist atropine. No receptor diminution was detected when the number of binding sites was evaluated with the lipophilic antagonist [³H]quinuclidinyl benzilate. This indicates that sodium nitroprusside induces a redistribution of the muscarinic receptors between the plasma membrane and an internal compartment of the cell. Receptor loss was readily reversed by treatment with the sulfhydryl reducing agent diethyldithiocarbamate. Our data provide evidence that muscarinic receptors are internalized by sodium nitroprusside through the oxidation of sulfhydryl groups; they also suggest that NO could play a role in muscarinic receptor desensitization.     

Interactions of quinidine and lidocaine with rat brain and heart muscarinic receptors

We have studied the effect of quinidine and lidocaine on binding to rat brain and cardiac muscarinic receptors. Both drugs had a higher affinity to brain stem and cardiac receptors, as compared with cerebral cortex, coinciding with the distribution of high-affinity agonist binding sites in the above tissues. The effects of the drugs on muscarinic antagonist and agonist binding did not fit simple competition to one receptor site, suggesting either preferential binding to high affinity agonist binding sites, or allosteric interactions. Batrachotoxin, which opens voltage sensitive sodium channels, had an opposite effect on agonist binding. The possibility of allosteric interactions between the muscarinic receptors and a site analogous to the sodium channel is discussed.     

Insertional mutagenesis of the arginine cage domain of the gonadotropin-releasing hormone receptor

The pattern of side-chain conservation at the cytoplasmic side of the third transmembrane domain of rhodopsin family G protein-coupled receptors, Asp/Glu-Arg-Tyr/X-X-X-Ile/Val, defines a structural "arginine cage" domain. Previous computational and mutagenesis studies of the GnRH receptor indicated an important contribution of local interactions to the function of this domain. We have investigated the functional importance of the intrahelical position and orientation of the arginine cage using insertional mutagenesis. Introduction of a single Ala proximal to the conserved Asp-Arg of this domain caused loss of detectable ligand binding. Inserting a second Ala, however, restored high-affinity agonist binding. Further insertion of three or four Ala residues at this site generated receptors that bound agonist with an affinity 3- to 10-fold higher than that of the wild-type receptor. Loss of detectable coupling to inositol phosphate turnover in all these mutant receptors confirms that the structure required in this region for efficient signaling is highly constrained. In contrast, the recovery of agonist binding with the progressive insertion of two to four Ala residues indicates that specific orientations of this segment can stabilize high-affinity receptor conformations that are uncoupled from signal transduction.     

Effects of inactivation of oxytocin receptor and inhibition of prostaglandin synthesis on uterine oxytocin receptor and gap junction formation and labor in the rat.

Normal term labor is associated with a surge in myometrial oxytocin receptor formation and gap junction development. We have previously shown that inhibition of prostaglandin synthesis by naproxen sodium, 2.0 mg/day, suppressed oxytocin receptor formation but not gap junction formation and prolonged gestation. In this study, we investigated the effects of a specific oxytocin antagonist on oxytocin receptor formation, gap junction formation, and labor in the rat. [Pen1,Phe(Me)2,Thr4,Orn8]oxytocin, a specific oxytocin antagonist, was infused subcutaneously during the last 3 days of pregnancy at 300 micrograms/day. Measurements of myometrial oxytocin receptor concentrations and gap junction formation on days 21 and 22 and days 22-23 (in labor) pregnant uteri showed no significant differences in the Bmax and Kd values between the control and the treated group. Gestation period was not prolonged by the oxytocin antagonist. However, in a separate group of day 23 pregnant rats, the uterine contractile response to 60 mU of oxytocin i.v. was found completely blocked by 10 micrograms of the oxytocin antagonist. These findings suggest that although functional oxytocin receptors did not appear to be essential for the initiation of labor, oxytocin antagonists may still be effective in the prevention of premature contractions. We also examined the effects of a higher dose of naproxen sodium, 5.0 mg/day, on gap junction formation. At this dose, naproxen sodium suppressed both oxytocin receptor and gap junction formation, prolonged gestation, and delayed parturition by 24 h or longer. Prostaglandin appears to be an important regulator or mediator of oxytocin receptor and gap junction formation and plays a critical role in the initiation of labor.     

Structural requirements for V2 vasopressin receptor proteolytic cleavage.

The ligand-induced proteolytic cleavage of the V2 vasopressin receptor transiently expressed in COS cells was investigated. After incubation of the cell membranes with a photoreactive ligand possessing full agonistic properties for V2 receptors, approximately 90% of the porcine and bovine V2 vasopressin receptors were cleaved in the upper part of transmembrane helix 2 at a heptapeptide sequence conserved in both vasopressin and oxytocin receptors. The oxytocin receptor was completely resistant to proteolysis after binding the same photoreactive ligand, which is only a partial agonist for this receptor. Chimeric V2/oxytocin receptors obtained by transfer of extracellular domains of the oxytocin receptor into the V2 receptor showed an increase in binding affinity for oxytocin versus vasopressin and a diminished cleavage. The proteolysis-resistant chimeric V2/oxytocin receptor, which contains the first three extracellular domains of the oxytocin receptor, stimulated cAMP accumulation to a larger extent in response to vasopressin than the wild-type receptor and showed impaired desensitization of the adenylate cyclase system. Our data indicate that the proteolytic cleavage of the V2 receptor requires a defined conformation, especially of the first two extracellular domains that is induced by agonist binding. Furthermore, the results suggest that the proteolytic V2 receptor cleavage might play a role in signal termination at elevated hormone concentrations.     

Determinants of high-affinity binding and receptor activation in the N-terminus of CCL-19 (MIP-3 beta)

CC chemokine receptor 7 (CCR-7) is expressed on mature dendritic cells and T-cells. Its ligands, CCL-19 (MIP-3beta) and CCL-21 (SLC), play an important role in the migration of these cells to secondary lymphoid organs where they are predominantly expressed. For most chemokines, the N-terminal domain preceding the first two conserved cysteines is involved in stabilizing the active conformation of its cognate receptors. We have chemically synthesized N-terminal analogues of CCL-19 with the aid of a native chemical ligation method to investigate structure function requirements of this ligand domain by performing ligand binding, GTP-gammaS binding, and chemotaxis assays. Successive truncations of the N-terminus of CCL-19 reduced the affinity of the receptor for the ligand in a size-dependent manner. Furthermore, Ala substitutions of Asn(3), Asp(4), and Asp(7) show that the side chains of these residues are important for high-affinity binding of CCL-19 to CCR-7. The effects observed were mirrored in both GTP-gammaS binding and chemotaxis assays, highlighting the functional importance of this ligand domain. We also describe two partial agonists of CCR-7 ([Nle(72)]CCL-19(6-77) and Ac-[Nle(72)]CCL-19(7-77)), and identify the first analogue of CCL-19 (Ac-[Nle(72)]CCL-19(8-77)) that acts as a functional antagonist in vitro (K(B) approximately 350 nM for GTP-gammaS binding assays). As mutations of both Glu(6) and Asp(7) to Ala did not dissociate effects on ligand binding from receptor activation, it is likely that the backbone of these two residues is crucial for agonist activity.     

Luteinizing hormone receptors in ovaries of the cynomolgus monkey (Macaca fascicularis) during the menstrual cycle

Ovarian luteinizing hormone (LH) receptors were characterized using ovarian tissues from 17 cynomolgus monkeys at different phases of the menstrual cycle. Low binding affinity receptors for ¹²⁵I-LH were observed throughout the menstrual cycle. The binding affinity of these receptors for LH (< 12 × 10¹⁰ M^?1) was approximately the same as that of ovarian LH receptors previously reported in human and nonhuman primates. In addition, high-affinity receptors (17?85 × 10¹⁰ M^?1) were also detected at the mid-luteal phase, during which a large functional corpus luteum was present. Thus the high-affinity LH receptors appear with the formation of the corpus luteum and disappear with its regression. Almost no fluctuation of binding capacity was observed throughout the menstrual cycle (32?112 fmol/ mg of ovarian tissue). The high-affinity LH receptor was judged to be derived from the functional corpus luteum.