Biomedical subjects

D Puett

Publications and source records attributed to D Puett.

At least 37 records · Page 2Linked to original sources

PubMed1997

Metabolism of endothelin-1 by neuroblastoma x glioma hybrid (NG108-15) cells.

The endothelin (ET) peptides, ET-1, ET-2, and ET-3, as well as the ETA and ETB receptor subtypes, are known to occur in brain, but there is a dearth of information on the metabolism of these peptides by the central nervous system (CNS). In this study we have investigated the kinetics of ET-1 binding to and dissociation from the hybrid neuroblastoma x glioma cell line, NG108-15, which is known to contain functional ET receptors, and metabolism of bound ET-1. [125I]ET-1 was incubated with cells for various periods of time up to 6 h, and the nature of the radioactivity in the cell medium and lysate was analyzed by reverse phase high performance liquid chromatography (HPLC). It was found that NG108-15 cells are capable of degrading [125I]ET-1 to [125I]Tyr and several fragments of intermediate hydrophobicity; however, a portion of the cell-associated [125I]ET-1 was protected from degradation for several hours.

Central Nervous System↗

PubMed1997

Circular dichroic spectroscopy of N-acetylglucosaminyltransferase V and its substrate interactions.

beta-1,6-N-Acetylglucosaminyltransferase V (EC 2.4.1.155) catalyzes the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc in beta(1,6)-linkage to the alpha(1,6)-linked mannose of N-linked oligosaccharides. Circular dichroism (CD) was used to investigate the secondary structure of a recombinant, soluble form of the enzyme and its interaction with UDP-GlcNAc and an inhibitory substrate analog. The CD spectrum of the apoenzyme indicated the presence of small amounts of beta-structure and substantial amounts (>50%) of alpha-helicity. The CD spectra of solutions containing UDP-GlcNAc and different ratios of UDP-GlcNAc:enzyme were measured. Interestingly, the spectrum of each mixture could not be accounted for by simple additivity of the two individual spectra, indicating a change in environment of the chromophores and/or a conformational change of the substrate or protein concomitant with binding. Similar results were obtained with mixtures of UDP and the enzyme. Analysis of the CD difference spectra at three wavelengths yielded an estimated average Kd of 4.4 mM for UDP-GlcNAc and 3.8 mM for UDP. By contrast, addition of the CD spectrum of an inhibitory substrate analog of its oligosaccharide acceptor substrate and the CD spectrum of the enzyme could account for that observed of an inhibitor-enzyme mixture; moreover, addition of the inhibitor to a mixture of UDP-GlcNAc and enzyme did not alter the Kd associated with UDP-GlcNAc binding to the enzyme. These results and kinetic studies reported herein suggest an ordered reaction in which UDP-GlcNAc binds first to the enzyme, followed by the sequential binding of the trisaccharide substrate.

Carbohydrate Sequence↗

PubMed1997

Identification of endothelin receptor subtypes in sheep choroid plexus.

Endothelin (ET) and its G-protein-coupled receptors are distributed in a wide variety of tissues, including the brain. In this study, we have identified and characterized the endothelin receptor subtypes in sheep choroid plexus. Competitive binding experiments using [125I]ET-1 and the receptor subtype-selective ligands, ET-1, ET-3, BQ-123, Sarafotoxin 6c, and [Ala1,3,11,15] ET-1 demonstrated the presence of both ETA and ETB receptor subtypes in the ratio of 30:70. In addition, a small fraction of the total binding sites exhibited affinities for ET-1 in the subpicomolar range. Chemical crosslinking of [125I]ET-1 with bis(sulfosuccinimidyl)-suberate (BS3) to choroid plexus membranes revealed the presence of two bands, with apparent molecular masses of 89 and 45 kDa, corresponding to the ETA receptor, and three bands, with apparent molecular masses of 75, 58, and 33 kDa, corresponding to the ETB receptor. Of considerable interest was the finding that dimers of the [125I]ET-1-occupied ETA receptor could be identified by crosslinking, as could apparent dimers and tetramers of [125I]ET-1, but only when bound to receptor. In addition to mapping the distribution of ET receptors in sheep choroid plexus, our results strongly suggest that ET-1 binding to the ETA receptor leads to dimer formation.

Animals↗

PubMed1997

Homodimer formation by the individual subunits of bovine lutropin as determined by sedimentation equilibrium.

Although differing in their amino acid sequences, the folding patterns of the alpha and beta subunits of human choriogonadotropin are similar in the crystal structure of the HF-treated glycoprotein hormone. Each subunit forms a cystine-knot motif like that found in several growth factors that form homodimers and heterodimers. In order to ascertain if the alpha and beta subunits can self-associate, e.g. to form homodimers, sedimentation equilibrium at various glycoprotein concentrations and temperatures was used to study the subunits of bovine lutropin, which are expected to exhibit conformations like those of the choriogonadotropin subunits. Each subunit was found to form homodimers with Kd values of 0.3 and 0.1 mM for alpha and beta respectively at 37 degrees C. Self-association was weakly exothermic for alpha and endothermic for beta; entropic factors made a major contribution for each. It is unlikely that homodimer formation of either subunit would be physiologically important, although homodimers may form to some extent intracellularly because of the relatively high concentrations during biosynthesis.

Animals↗

PubMed1996

hCG-receptor binding and transmembrane signaling.

The technique of site-directed mutagenesis has proven to be quite powerful in elucidating contact sites involved in the interaction of the heterodimeric glycoprotein hormones and their respective seven transmembrane (TM) G protein-coupled receptors. Our laboratory has focused on identification of the minimum core sequences of the alpha and beta subunits required for bioactivity, the minimum length of a conjoined (yoked) single-chain hCG, the amino acid residues on hCG and the LH/CG-receptor (LH/CG-R) responsible for high-affinity binding, and the regions of the receptor that are involved in TM signaling. A number of amino acid residues have been mapped on the alpha and beta subunits of hCG that appear important in receptor binding. When projected onto the crystal structure of HF-treated hCG, these residues, by and large, cluster on one side of the molecule and cover a sizeable surface area, indicating that the hormone-receptor binding interface is rather extensive. Based on mutagenesis studies of several conserved ionizable amino acid residues in the extracellular domain (ECD) of LH/CG-R and a model that we, in collaboration with Drs Lapthorn and Isaacs, have developed for this region based on the crystal structure of porcine ribonuclease inhibitor, a charged region that appears to play an important role in hormone-receptor recognition has been identified. We have also delineated several regions of LH/CG-R that do not appear to participate in hCG binding but are involved in hCG-mediated signaling. These regions are located in the ECD and extracellular loop III just prior to entry into the membrane via TM helices I and VII, respectively, and in TM helices VI and VII. Similarly, a homologous region in the ECD of the FSH receptor, located with ten residues of TM helix I, is important in signaling but not hormone binding. These results suggest that ligand binding and ligand-mediated receptor activation are quasi-distinct, albeit sequential phenomena. Collectively, our mutagenesis and modeling studies, coupled with results from other laboratories, argue for a ligand-induced conformational change of the receptor that may involve a relative reorientation of the TM helices.

Amino Acid Sequence↗

PubMed1996

Protein engineering of a novel constitutively active hormone-receptor complex.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone consisting of an alpha and a beta subunit that stimulates intracellular levels of cAMP via a G protein-coupled receptor. Herein we report the engineering and characterization of a novel molecule in which the receptor and its heterodimeric ligand were covalently linked in a single polypeptide chain. The hormone-receptor complex was expressed in cells transfected with this construct, but the cells were unable to bind significant amounts of exogenous hCG. However, cleavage of the hormone with a site-specific protease rendered the receptor accessible to exogenously added hormone. Cells transfected with the hCG-receptor construct contained elevated basal levels of cAMP; moreover, addition of hormone had no significant effect. These results are consistent with a strong and stable interaction between the single-chain hormone and its covalently linked receptor that results in a constitutively active complex.

Animals↗

PubMed1996

Endothelin degradation by vascular smooth muscle cells.

The mechanism(s) of degradation of the potent vasoconstrictor endothelin-1 (ET-1) by rat vascular smooth muscle A-10 cells, which possess the ETA receptor subtype, was investigated by incubating [125I]ET-1 (0.1 nM) with cells for 0-4 h at 37 degrees C in the presence and absence of lysosomal enzyme inhibitors, NH4Cl and chloroquine, and a neutral endopeptidase inhibitor, phosphoramidon. The assay buffer and cell extracts were analyzed by reverse-phase HPLC, and the radioactivity in the fractions was measured. In the absence of inhibitors, most of the radioactivity in the medium was in the form of [125I]Tyr after a 4 h incubation. When [125I]ET-1 was incubated with A-10 cells at 4 degrees C, six radiolabeled peaks, including some [125I]Tyr and about 30% of the original [125I]ET-1, were present in the medium. In the presence of 5 microM chloroquine there was no [125I]Tyr peak in the medium, indicating that internalization and putative lysosomal degradation of ET-1 were blocked. NH4Cl (50 and 100 mM) also reduced the amount of [125I]Tyr formed. The presence of ET-1 fragments indicated that, in addition to lysosomal degradation, some of the ligand is metabolized by enzymes located on the cell membrane; we demonstrated, however, that secreted proteases from A-10 cells are not involved in the degradation of ET-1. The neutral endopeptidase inhibitor, phosphoramidon, did not completely inhibit the metabolism of [125I]ET-1 to [125I]Tyr. These results establish that various cell-associated enzymes are capable of degrading ET-1 in A-10 cells. Moreover, analysis of the cell lysates indicated the presence of a relatively stable pool of ET-1-occupied receptors or compartmentalized ET-1, protected from cell proteases, which may contribute to the potent contractility of ET-1.

Animals↗

PubMed1996

Identification of conventional and novel endothelin receptors in sheep choroid plexus cells.

Previous studies have demonstrated the presence of super-high affinity endothelin receptors with apparent Kd's on the order of pM in different brain tissues. This study was designed to characterize, in detail, the receptors present in SCP cells, a non-transformed sheep choroid plexus cell line. Competitive binding assays with receptor-selective ligands indicated the presence of at least three classes of binding sites: a conventional receptor of the ETA subtype with a Kd = 0.4 nM that mediates an increase in intracellular levels of inositol 1,4,5-trisphosphate (IP3)in response to ET-1 and two additional sites with much higher binding affinities. The latter two sites are not coupled to the common signal transduction pathways of IP3, cAMP and cGMP. Northern blot analysis confirmed the presence of only the ETA subtype mRNA in SCP cells. It remains to determined if the multiple binding sites are distinct gene products, multiple affinity states of a single receptor molecule or a result of cooperative association of one site with either the ligand or with other proteins.

Animals↗

PubMed1996

Identification of amino acid residues in transmembrane helices VI and VII of the lutropin/choriogonadotropin receptor involved in signaling.

The lutropin/chroriogonadotropin receptor (LH/CG-R) is a member of the G protein-coupled receptor superfamily containing a relatively large extracellular domain responsible for high affinity ligand binding. There is a paucity of information on the mechanism of ligand-mediated transmembrane signaling via the seven transmembrane helices (TMH) of these receptors. In the present study we have used site-directed mutagenesis to replace each of nine conserved amino acid residues in the TMHs of rat LH/CG-R. COS-7 cells were transiently transfected with the eukaryotic expression vector pSVL-LH/CG-R, wild-type and mutants, followed by measurements of human CG binding and human CG-mediated cAMP production. Three point mutants of LH/CG-R were prepared that diminished signaling but not binding: Pro-562-Leu (TMH VI), Pro-591-Leu(TMH VII), and Tyr-601-Ala (TMH VII). Two point mutants of LH/CG-R, Pro-479-Leu(TMH IV) and Pro-598-Leu(TMH VII), resulted in impaired localization, and four receptor mutants, Thr-424-Ala (TMH III), Ser-562-Ala (TMH VI), Met-560-Leu (TMH VI), and Tyr-590-Ala (TMH VII), were similar to wild-type LH/CG-R. In summary, these findings indicate a critical role of Tyr-601 in transmembrane signaling of LH/CG-R since an Ala replacement results in almost total abolition of cAMP production in response to human CG; prolines 562 and 591 also appear to be important for full signaling.

Amino Acid Sequence↗

PubMed1996

Expression of functional lutropin/choriogonadotropin receptor in the baculovirus system.

The LH/CG receptor (LH/CG-R) is a G protein-coupled receptor with a relatively large glycosylated extracellular domain. The complete 674 amino acid residue rat receptor was expressed in Sf9 insect cells using the baculovirus expression system. Optimal expression under the control of the polyhedrin promoter was obtained at 72 h post-infection and a multiplicity of infection of 0.1. The recombinant LH/CG-R was expressed on the cell surface (ca. 4500 receptors/cell) and exhibited saturable, high affinity binding of human CG (hCG) with a Kd of 0.4 nM. There was no evidence of intracellular trapping of the receptor. The intracellular concentration of cAMP was increased in response to hCG binding. In contrast, baculovirus-expressed recombinant hCG only weakly stimulated intracellular cAMP levels at relatively high doses. Two forms of the receptor (approximately 75 and approximately 200 kDa) were detected by Western blot analysis. These results demonstrate that the full length LH/CG-R expressed in insect cells is functional in that it binds hormone with high affinity and is able to couple to adenylate cylase.

Adenylyl Cyclases↗

PubMed1996

Lys583 in the third extracellular loop of the lutropin/choriogonadotropin receptor is critical for signaling.

The lutropin/choriogonadotropin receptor (LH/CG-R) contains a relatively large extracellular domain, in addition to the seven transmembrane helices (TMH), three extracellular loops (ECL), and three intracellular loops typical of G protein-coupled receptors. While high affinity ligand binding has been attributed to the N-terminal extracellular domain, there is evidence that portions of the three ECLs may function in ligand binding and transmembrane signaling. We have investigated the role of several ionizable amino acid residues of rat LH/CG-R in human choriogonadotropin (hCG) binding and hCG-mediated cAMP production. COS-7 cells were transfected with the pSVL expression vector containing cDNAs of either wild-type or mutant rat LH/CG-R. Several point mutants of Lys583, located at the interface of ECL III and TMH VII, bound hCG like wild-type receptor but exhibited greatly diminished ligand-mediated signaling. Neither the point mutant, Lys401-->Asp (ECL I), nor the double mutant, Asp397-->Lys/Lys583-->Asp (ECLs I and III, respectively, showed significant hCG binding to intact cells; in detergent-solubilized cells, only the double mutant bound hCG. The mutants Arg341-->Glu (interface of the extracellular domain and TMH I) and Lys488-->Glu (ECL II) proved to be similar to wild-type receptor in binding and signaling. Our results establish that Lys583 is important in signaling but not ligand binding. Its location on the opposite side of the membrane from GS precludes a direct interaction, thus emphasizing the importance of a conformational change in the receptor and suggesting that ligand binding to receptor and ligand-mediated receptor activation are dissociable phenomena.

Amino Acid Sequence↗

PubMed1996

Circular dichroic spectroscopy of Arg46-nicked ovine lutropin alpha and derived fragments.

The alpha subunit of ovine lutropin can be nicked with the endoproteinase Arg-C to give a single cleavage of the Arg46-Ser47 peptide bond. Following reduction by sulfitolysis, the N-terminal (residues 1-46) and C-terminal (residues 47-96) fragments can be separated and then recombined and reoxidized to yield a reconstituted nicked alpha that binds to the beta subunit but exhibits only 2-3% of the receptor-binding potency of intact lutropin. We have investigated nicked alpha, the two separated fragments, and reconstituted nicked alpha by circular dichroic spectroscopy and compared the spectra with those of intact alpha and reduced, reoxidized intact alpha. Between 200 and 225 nm the spectra of the two intact preparations are similar, as are the spectra of the two nicked preparations. However, the extremum negative ellipticities of the nicked preparations are substantially less than those of the intact preparations between 210 and 220 nm, indicating a loss in secondary structure accompanying cleavage of the Arg46-Ser47 bond. The sum of the spectra of the two fragments is significantly different from that of reconstituted nicked alpha, showing that the secondary structures in the isolated fragments are quite different from that of the reconstituted nicked protein. Reduced receptor binding by lutropin preparations containing a nicked alpha subunit may be attributable in part to the loss of secondary structure, probably helicity.

Animals↗

PubMed1996

Racial differences in plasma endothelin-1 concentrations in individuals with essential hypertension.

Hypertension is more prevalent in blacks than whites, and the reasons for this difference remain unclear. To test whether endothelin may play a role in these racial variations, we analyzed plasma samples from black and white women and men with high blood pressure by an enzyme-linked immunoassay specific for endothelin-1 (ET-1), a potent vasoconstrictor, and compared them with those obtained from similar subjects with normal blood pressure. Both female and male hypertensive blacks had elevated levels of immunoreactive ET-1 (11.3 +/- 1.0 and 12.3 +/- 1.3 pmol/L, respectively) compared with values in normotensive control blacks (1.5 +/- 0.2 and 1.4 +/- 0.2 pmol/L). Corresponding values in female and male hypertensive whites were 3.8 +/- 0.6 and 3.8 +/- 0.6 pmol/L, respectively, compared with respective values of 1.4 +/- 0.1 and 2.8 +/- 0.4 pmol/L in normotensive control whites. These results indicate that plasma concentrations of immunoreactive ET-1 levels differ significantly between black and white individuals with high blood pressure. This finding may be an important factor in the etiology of racial differences in the prevalence and severity of hypertension and deserves further study [corrected].

Adult↗

PubMed1996

Site-directed mutagenesis of amino acids 33-44 of the common alpha-subunit reveals different structural requirements for heterodimer expression among the glycoprotein hormones and suggests that cyclic adenosine 3',5'-monophosphate production and growth promotion are potentially dissociable functions of human thyrotropin.

Amino acid residues 33-44 of the common alpha-subunit of the glycoprotein hormones have been implicated in heterodimerization as well as high affinity receptor binding of human (h) CG. In the present study, we compared the role of specific amino acids within this region for glycoprotein hormone heterodimer formation, using a transient transfection system to coexpress different mutant alpha-subunit constructs with the beta-subunit of either hTSH, hCG, or hFSH. Our results identified a crucial role for alpha Pro38 in the heterodimer expression of hTSH as well as hFSH, similiar to what had been described for hCG. In contrast, alpha Ala38, which had been critical for hCG, was not essential for hTSH heterodimer expression and less important for hFSH, whereas alpha Phe33 and alpha Arg35 appeared uniquely important for hFSH. Furthermore, we assessed the role of these residues for bioactivity and receptor binding of hTSH. Mutation of the surface-exposed residues alpha Arg42-Ser43-Lys44, which form part of a unique alpha-helical structure, to Ala42-Ala43-Ala44, decreased TSH receptor binding using porcine thyroid membranes as well as rat FRTL-5 cells. Residues alpha Phe33 and alpha Arg35, in contrast, were not important for high affinity binding of hTSH. In the signal transduction of hTSH, alpha Ala36 was necessary for efficient growth induction in FRTL-5 cells but not for cAMP production in either FRTL-5 cells or Chinese hamster ovary cells expressing the human TSH receptor (JP09). Similarly, residues alpha Arg42-Ser43-Lys44 were more important for hTSH-mediated induction of cell growth than cAMP production. Mutating alpha Arg35 to Ala reduced cAMP induction but not receptor binding of hTSH. In summary, using site-directed mutagenesis, we identified a domain, residues 33-44 of the common alpha-subunit, important in heterodimer expression, receptor binding, and activation of hTSH. The comparison of the relative roles of specific amino acids within this region in hTSH with hCG and hFSH highlights previously unrecognized differences in the structural requirements for heterodimer expression among the members of the glycoprotein hormone family. Moreover, our findings revealed a novel role for residues alpha 33-44 in triggering different postreceptor events, suggesting that cAMP production and growth promotion may, at least in part, be dissociable functions of hTSH.

Amino Acid Sequence↗

PubMed1996

Determination of residues important in hormone binding to the extracellular domain of the luteinizing hormone/chorionic gonadotropin receptor by site-directed mutagenesis and modeling.

The LH/CG receptor (LH/CG-R) belongs to the family of glycoprotein hormone G protein-coupled receptors. The extracellular domain of LH/CG-R is associated with high ligand-binding affinity and contains leucine-rich repeats (LRRs). With the goal of identifying essential amino acid residues involved in ligand binding, we replaced several conserved ionizable residues in the rat LH/CG-R with ones of opposite charge. The expression of these mutants was assessed by binding studies and Western blots after COS-7 cells were transiently transfected with wild type and mutant receptor cDNAs. The charge inversion of each of Lys40, Lys104, Asp118, Glu132, and Asp135 with Asp or Lys resulted in no detectable human CG binding in intact or solubilized cells; as control, a Lys40-->Arg replacement yielded a mutant with characteristics of the wild type receptor. Western analysis showed that the Lys40-->Arg mutant expressed at a level comparable to that of wild type receptor and, like wild type, exhibited a predominant immunoreactive mature form of LH/CG-R. Each of the five charge inversion mutants expressed at a lower level than wild type as assessed by immunoreactivity, and the levels of the Lys40-->Asp and Glu132-->Lys mutants were particularly low. The ratio of the mature to immature form of the receptor was high, i.e. like that of wild type, for the Glu132-->Lys and Asp135-->Lys replacements; the three other charge inversion mutants exhibited less mature than immature forms of the receptor. To aid in interpreting these results, we developed a model incorporating residues 27-235 of the extracellular domain of the rat LH/CG-R based on the crystal structure of the porcine ribonuclease inhibitor. Sequence homology and alignment revealed nine LRRs, with flanking cysteine clusters as found in a number of LRR proteins. Our model suggested that the Lys replacements of Glu132 and Asp135, i.e. those mutants that formed mature receptors, would disrupt the regional negative charge of the receptor. We propose that these residues are either directly involved in hormone binding or indirectly by disruption of the charge of an important binding surface.

Amino Acid Sequence↗

PubMed1996

Mutations of Arg68 of the human chorionic gonadotrophin beta subunit lead to reduced secretion.

The Arg68-Leu69 sequence is invariant in the beta subunits of chorionic gonadotrophin and luteinizing hormone from a variety of species. Using site-directed mutagenesis of the human chorionic gonadotrophin (hCG)-beta cDNA, several replacements of Arg68, an Ala replacement of Leu69, and a multiple replacement with Ala-Ala-Ala-Ala of the tetrapeptide sequence, Arg68-Leu69-Pro70-Gly71, were prepared and characterized. The wild-type and mutant cDNAs were subcloned into a pRSV expression vector and transiently transfected into CHO cells containing a stably integrated gene for bovine a. Concentrations of secreted wild-type and mutant hCG-beta subunit and holoprotein were determined using radioimmunoassays; potencies, i.e. the ratio of biologic to immunologic activity, of several of the mutant heterodimers were measured in vitro via gonadotrophin-mediated steroidogenesis in transformed murine Leydig cells (MA-10). The Leu69-->Ala mutant formed a mutant holoprotein that was essentially equipotent with wild-type hormone in the steroidogenesis assay. The Arg68 replacements with Lys, Ala, and Leu were poorly secreted by the cells, e.g. < 10% that of wild-type hCG; however, sufficient quantities of mutant holoproteins containing Lys68 and Ala68 were obtained for biological assays, and both exhibited greater apparent potencies than wild-type hormone. Likewise, a mutant holoprotein containing the Arg68-Leu69-Pro70-Gly71-->Ala-Ala-Ala-Ala multiple replacement was apparently more potent than wild-type hormone, but it too was secreted at lower levels than wild-type. These results establish that replacements of Arg68 in hCG-beta diminish secretion, but the small amount of holoprotein that is formed and secreted appears to be of somewhat greater potency than wild-type hormone.

Animals↗

PubMed1996

Endothelin binding to NG108-15 cells: evidence for conventional ETA and ETB receptor subtypes and super-high affinity binding components.

The endothelin (ET) peptides have been identified in the CNS, but there is a paucity of information on their physiological roles. NG108-15 cells, a clonal strain of a neuroblastoma x glioma hybrid cell line, have been widely used in neurobiological research since they retain certain differentiated properties of the non-transformed parental cells. It is known that NG108-15 cells respond to the ET peptides, but only limited information is available on the characterization of the ET receptors that mediate these effects. The present study was designed to identify the type(s) of ET receptors on NG108-15 cells in a proliferative state by competitive binding assays using [125I]ET-1 as the radiolabelled ligand and the receptor-selective ligands. ET-1, ET-3, BQ-123, sarafatoxin-6-c and [Ala1,3,11,15]ET-1. The results suggested the presence of conventional ETA and ETB receptor subtypes, with ETA in excess over ETB. These findings were consistent with the results of Northern analysis in that mRNAs encoding the ETA and ETB receptor subtypes were identified in NG108-15 cells, with a preponderance of ETA to ETB. Of considerable interest was the observation of other ET-binding components with much higher affinities than the conventional receptors. It remains to be demonstrated if these particular binding components are functional and represent differ gene products or arise from association of the conventional ETA and ETB receptor subtypes with themselves or other structures, e.g. proteins or lipids, of CNS origin.

Animals↗

PubMed1995

Identification of two amino acid residues on the extracellular domain of the lutropin/choriogonadotropin receptor important in signaling.

The lutropin/choriogonadotropin receptor (LH/CG-R) is a G protein-coupled receptor with a relatively large extracellular domain. The cDNAs of LH/CG-R wild type and 15 point and double mutants, which encoded residues of opposite charge to that of wild type, were transiently transfected into COS-7 cells. Human choriogonadotropin (hCG) binding was determined, as was hCG-mediated cAMP production. Most of the replacements resulted in no substantive effect on the binding affinity of hCG to LH/CG-R or on hCG-stimulated cAMP production, although the mutants expressed at a lower level than LH/CG-R wild type. The most interesting observation was noted with two point mutants of LH/CG-R, Glu332-->Lys and Asp333-->Lys, which bound hCG but failed to give increased cAMP production. Several of the mutant forms of LH/CG-R that expressed at low levels were further analyzed by soluble binding assays and Western blots. There was no evidence of any significant degree of intracellular trapping of hCG-binding mutant receptors. The expected major (93 kDa) and minor (78 kDa) forms were found for LH/CG-R wild type and several of the mutants. The Lys235-->Asp and Asp333-->Lys mutants exhibited primarily the lower M(r) form, indicating that receptor processing was impaired or that the mutant higher M(r) form was more rapidly degraded than LH/CG-R wild type. These results demonstrate that Glu332 and Asp333, which are located near the first transmembrane helix, are important in receptor activation, while other conserved ionizable residues of LH/CG-R appear important in cell surface expression or stability but not in binding or signaling.

Amino Acid Sequence↗