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Query: UNIPROT:P06889 (Mol)
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During the molecular cloning of the ovine testicular follicle-stimulating (FSH) receptor that couples to the Gs-type effector systems, we discovered novel cDNA clones that were highly homologous. Some of these clones contained an insert of 1,584 bp, which consisted of a divergent 3' region spliced with a 5' region that was identical to nucleotides 724-1,924, forming part of the 9th and 10th exons, of the coding region of the ovine FSH receptor gene. The prominence of alternately spliced clone, which suggested important functional implications, prompted this detailed investigation. Screening of the library by polymerase chain reaction and Northern analysis of testicular messenger RNA with a specific ribo-probe directed to the divergent 3' region of this transcript suggested existence of a full-length transcript of roughly 2.4 kb size. The cDNA was assembled and characterized for its structure. The predicted full-length sequence consisting of nucleotides -121-1,924 of the ovine FSH receptor and the novel 3' region (nucleotides 1,925-2,307) encoded a protein of 670 amino acids containing the entire extracellular and transmembrane domains of the ovine FSH receptor. However, a frame-shift in the coding sequence at the point of divergence resulted in a shorter (40 residues vs. 65 for ovine FSH receptor) C-terminus with three cysteine residues and a reduced number of potential phosphorylation sites. Two of the cysteine residues were adjacent and are apparently potential double palmitoylation sites compared to the single site present in the Gs coupled ovine FSH receptor. Stable expression of this novel transcript in human embryonic kidney (HEK 293) cells revealed the complete absence of cyclic AMP inducible functions, but presence of specific hormone binding activity on plasma membranes and prominent cell surface immunostaining by antireceptor antiserum. There was no alteration in hormone binding specificity because the structurally analogous luteinizing hormone (LH) did not bind to the receptor. The loss of cyclic AMP stimulation in the transfected cells was completely opposite to the properties of the cells expressing the active wild-type receptor. When cells expressing active receptors were cotransfected with the altered FSH receptor cDNA, hormone action was inhibited, suggesting that it could be functioning as a dominant negative receptor. The existence of this ovine FSH receptor with an altered carboxyl terminus predicts the utilization of an alternative splicing mechanism for regulation of receptor expression, signalling and gonadal function. Our study reveals that the modular structure of the FSH receptor gene generates motifs that allows coupling to different effectors. This could become a common feature for all glycoprotein hormone receptors.
Mol Reprod Dev 1997 Dec
PMID:Molecular cloning, structure, and expression of a testicular follitropin receptor with selective alteration in the carboxy terminus that affects signaling function. 936 40

Pituitary follitropin (FSH) has pleiotropic actions on gonads, but it is not certain if all these events are mediated by a single receptor. A single gene for the FSH receptor undergoes extensive alternate splicing generating multiple transcripts, and several of these have been cloned and characterized from the sheep testis. In this study we have investigated the expression in HEK (human embryonic kidney) 293 cells of a cloned cDNA encoding the first eight exons of the FSH receptor along with a carboxyterminal extension that contributed a hypothetical single transmembrane domain. This cDNA, which lacked the conventional seven transmembrane motif of the full-length 695 residue wild-type receptor protein, was also efficiently expressed on the cell surface and exhibited high affinity and specificity for FSH binding. LH did not compete for FSH binding indicating that these structures contained all the motifs necessary for specific hormone recognition. Following hormone binding and affinity crosslinking the deduced M(r) of the expressed receptor was compatible with dimer formation. The expression of these altered FSH receptors on the cell surface was confirmed by immunohistochemistry, which revealed punctate labeling in a pattern comparable to that shown by cells transfected by wild-type receptor cDNA. Addition of FSH stimulated 3H-thymidine incorporation in transfected cells in a biphasic manner. By performing RT-PCR we could show that similar altered receptor transcripts were present in both the ovary and testis. Our results reveal for the first time that the seven transmembrane structure of FSH-receptor is not absolutely necessary for cell surface expression and hormone binding provided other compensating motifs are present in the protein structure for membrane insertion. Some of these features are typical of growth factor receptors. Our investigations also demonstrate that alternate splicing of the FSH receptor gene provides a mechanism for creating receptor diversity and suggest that multiple receptors could be involved in regulation of hormone action.
Mol Reprod Dev 1997 Dec
PMID:Alternative splicing converts the G-protein coupled follitropin receptor gene into a growth factor type I receptor: implications for pleiotropic actions of the hormone. 936 41

Biochemical properties of mutant type 2 vasopressin receptors (V2Rs) causing a partial phenotype of nephrogenic diabetes insipidus were investigated in transiently transfected HEK 293 cells. Cell surface expression of the V2R was not altered by substituting Asp85 in the second transmembrane region by Asn as determined by saturation binding assays. Although the affinity of the mutant V2R for arginine vasopressin (AVP) was reduced only 6-fold, the response of adenylyl cyclase activity to AVP revealed a 50-fold right shift in EC50 and a decreased maximum response for the mutant V2R. These data indicated that replacement of Asp85 by Asn affected coupling of the receptor to Gs, a conclusion substantiated by a 20-fold decrease in the calculated coupling efficiency of this receptor. The Gly201Asp mutation in the second extracellular loop, also found associated with an NDI partial phenotype, decreased cell surface expression of the V2R with minor reduction in ligand-binding affinity and coupling efficiency to Gs. A pronounced difference was observed for this mutant V2R between the stimulation of adenylyl cyclase activity promoted by AVP and the V2 vasopressin receptor agonist deamino[Cys1,D-Arg8]-vasopressin, suggesting an involvement of Gly201 in the selectivity of the receptor for different ligands. These data demonstrated that while decreased ligand-binding affinity and decreased coupling to Gs are responsible for the attenuation of response to ligand in the Asp85Asn mutant V2R, cell surface expression of the V2R is the major factor reducing cellular responses to ligand for the Gly201Asp mutant V2R.
Mol Endocrinol 1997 Nov
PMID:Biochemical basis of partial nephrogenic diabetes insipidus phenotypes. 936 48

Human breast cell carcinoma MCF-7 cells were found to bind 125I-labeled rat amylin (rAmylin) and the peptide amylin antagonist radioligand 125I-AC512 with high affinity. This high affinity binding possessed characteristics unique to the already defined high affinity binding site for amylin in the rat nucleus accumbens [Mol. Pharmacol. 44:493-497 (1993); J. Pharmacol. Exp. Ther. 270:779-787 (1994); Eur. J. Pharmacol. 262:133-141 (1994)]. To further define this receptor, we report results of expression cloning studies from an MCF-7 cell library. We isolated two variants of a seven-transmembrane receptor that were identical to two previously described human calcitonin receptors (hCTR1 and hCTR2). These receptors were characterized by expression in different surrogate host cell systems. Transient expression of hCTR1 in COS cells yielded membranes that bound 125I-AC512 and 125I-salmon calcitonin with high affinity, but no high affinity binding was observed with 125I-human calcitonin (hCAL) or 125I-rAmylin. Stable expression of hCTR1 in HEK 293 cells produced similar data. In contrast, expression of hCTR2 in COS cells yielded membranes that bound 125I-AC512, 125I-hCAL, and 125I-rAmylin with high affinity. The agonists 125I-hCAL and 125I-rAmylin bound 65% and 1.5%, respectively, of the sites bound by the antagonist radioligand 125I-AC512 in this expression system. This pattern of binding was repeated in HEK 293 cells stably transfected with hCTR2 (125I-hCAL = 24.8% Bmax, 125I-rAmylin = 8% Bmax). In both expression systems, the agonists hCAL and rAmylin were much more potent in displacing their radioligand counterparts than was the antagonist radioligand 125I-AC512. For example, the pKi value for displacement of 125I-AC512 by rAmylin was 7.2 in HEK 293 cells but rose to 9.1 when displacing 125I-rAmylin. Finally, hCTR2 was expressed in baculovirus-infected Ti ni cells. In this system, only specific binding to the antagonist 125I-AC512 and agonist 125I-hCAL was observed; no binding to 125I-rAmylin could be detected. These data are discussed in terms of two working hypotheses. The first is that amylin is a weak agonist for hCTR2 and that this receptor is unrelated to the amylin receptor found in this cell line. The second is that hCTR2 couples to different G proteins for calcitonin and amylin function in different cells. At present, these data cannot be used to disprove conclusively either hypothesis.
Mol Pharmacol 1997 Dec
PMID:Expression cloning and receptor pharmacology of human calcitonin receptors from MCF-7 cells and their relationship to amylin receptors. 939 87

Differential immunohistochemical labeling is often observed using different antibodies against the same protein. Two polyclonal antipeptide antibodies against the 5-HT1A receptor have been generated by our group. The S1A-170 (aa 170-186) and 258 (aa 258-274) are specific for sites in the second extracellular loop and third intracellular loop, respectively [E.C. Azmtia, I. Yu, H.M. Akbari, N. Kheck, P.M. Whitaker-Azmitia and D.R. Marshak, Antipeptide antibodies against the 5-HT1A receptor, J. Chem. Neuroanat., 5 (1992) 289-298]. Comparison of the labeling patterns of these two antibodies and other antipeptide antibodies against the 5-HT1A receptor revealed that although similar populations of cells were labeled, individual antibodies favor certain staining patterns. Immunocytochemistry and western blotting results of transfected cell lines and brain tissue revealed the following: (1) both the S1A-170 and S1A-258 are specific for the 5-HT1A receptor when used for immunocytochemistry in transfected HEK-293 and COS-1 cells; (2) when expressed in cultured cell lines, the 5-HT1A receptor is differentially glycosylated dependent on cell type, and the S1A-258 is specific for only certain species on immunoblots; and (3) the S1A-258 and L5B7 [M. Riad, S. El Mestikawy, D. Derge, H. Gozlan, and M. Hamon, Visualization and quantification of central 5-HT1A receptors with specific antibodies, Neurochem. Int., 4 (1991) 413-423] label common bands at 40 and 70 kDa on immunoblots of hippocampal proteins, but show opposite staining intensities. These results provide evidence for the immunocytochemical specificity of both the S1A-170 and S1A-258 and suggest that the discrepancies noted in immunohistochemistry may be due in part to different molecular conformations.
Brain Res Mol Brain Res 1997 Oct 15
PMID:Molecular characterization of antipeptide antibodies against the 5-HT1A receptor: evidence for state-dependent antibody binding. 940 44

The effect of nicotine on the major human neuronal nicotinic receptor (alpha 4 beta 2 subtype) was studied in permanently transfected HEK 293 cells. Prolonged exposure to low concentrations of nicotine (1 microM) increased epibatidine binding but functionally deactivated the nicotinic receptor, abolishing Ca2+ influx in response to an acute nicotine challenge. Deactivation could also be caused by down-regulating protein kinase C (PKC) activity with 0.5 microM phorbol-12,13-dibutyrate or briefly incubating cells with the PKC inhibitor NPC-15437. Recovery from receptor deactivation caused by either nicotine treatment or PKC inhibition occurred slowly (4-6 hr). Reversal of nicotine-induced deactivation was accelerated by the addition of inhibitors of protein phosphatases 2A and 2B. These data suggest a hypothetical mechanism of nicotine-induced deactivation that involves dephosphorylation of nicotinic receptors at PKC phosphorylation sites.
Mol Pharmacol 1997 Dec
PMID:Functional deactivation of the major neuronal nicotinic receptor caused by nicotine and a protein kinase C-dependent mechanism. 941 21

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G-protein-coupled receptors. Through expression cloning, human and rat GALR1 receptor cDNA clones have previously been isolated and characterized. In this study, we have used homology screening to isolate a rat brain cDNA clone encoding a second galanin receptor subtype, the GALR2 receptor. The isolated cDNA encodes a 372-amino-acid G-protein-coupled receptor that shares 38% overall amino-acid identity with the rat GALR1 receptor. The pharmacological profile of the rat GALR2 receptor is similar to that of the rat GALR1 receptor. The rat GALR2 receptor binds galanin, N-terminal galanin fragments, and the putative galanin receptor antagonists galantide, C7, M35 and M40 with high affinity but it does not bind C-terminal galanin fragments. Galanin increases intracellular inositol phosphate levels in HEK 293 cells expressing the rat GALR2 receptor via a pertussis toxin-insensitive G-protein. The rat GALR2 receptor mRNA is highly expressed in several brain regions, including hypothalamus and hippocampus as well as the anterior pituitary, with lower levels of expression detected in amygdala, and regions of cortex. It is also highly expressed in the GH3 pituitary cell line and in gut tissues, and to a lower extent in spleen, lung, skeletal muscle, heart, kidney, liver and testis. These results suggest that GALR2 receptor mediates galanin's regulation of pituitary hormone secretion and possibly food intake.
Brain Res Mol Brain Res 1997 Nov
PMID:Cloning, pharmacological characterization and distribution of a novel galanin receptor. 942 6

Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the alpha2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative alpha2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the alpha3 and alpha4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B alpha2 helix mutants were not efficiently prenylated as determined by incorporation of [3H]mevalonate. Metabolic labeling studies using [32P]orthophosphate indicated that the poor prenylation of the Rab1B alpha2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the alpha2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate.
Mol Biol Cell 1998 Jan
PMID:The putative "switch 2" domain of the Ras-related GTPase, Rab1B, plays an essential role in the interaction with Rab escort protein. 943 2

Despite an intriguing cell biology and the suggestion of a role in pathophysiological responses, the mechanism of action of such lipid phosphoric acid mediators as lysophosphatidic acid (LPA) remains obscure, in part because of an underdeveloped medicinal chemistry. We report now the agonist activity of a synthetic phospholipid in which the glycerol backbone of LPA is replaced by L-serine. Like LPA, the L-serine-based lipid mobilizes calcium and inhibits activation of adenylyl cyclase in the human breast cancer cell line MDA MB231. Treatment with LPA desensitizes MDA MB231 cells to subsequent application of the L-serine compound; when the order of application is reversed, however, the L-serine compound does not prevent calcium mobilization by LPA, which might indicate the existence of two LPA receptors in these cells. The analogous D-serine-based phospholipid was distinctly less potent than the L-isomer in those assays; this finding demonstrates stereoselectivity by an LPA receptor. Unlike LPA, the L-serine-based lipid does not evoke a chloride conductance in Xenopus laevis oocytes, but injection of poly(A)+ RNA from HEK 293 cells confers this phenotype on the oocyte. The latter result has practical importance in that it allows use of the frog oocyte for expression cloning of an LPA receptor DNA, an assay system made problematic by the oocyte's strong endogenous response to LPA.
Mol Pharmacol 1998 Feb
PMID:Characterization of a receptor subtype-selective lysophosphatidic acid mimetic. 946 75

Polymerase chain reaction and rapid amplification of cDNA ends were used to isolate cDNAs encoding a 5-hydroxytryptamine3 (5-HT3) receptor subunit and its splice variants from guinea pig intestine. The amino acid sequence predicted from this cDNA is 81% homologous to the murine 5-HT3 receptor subunits cloned from NCB20 and N1E-115 cells. The splice variants code for two proteins differing by a deletion of six amino acids located in the large intracellular loop between transmembrane domains M3 and M4. For characterization, the cloned 5-HT3 cDNA was expressed in HEK 293 cells, and the electrophysiological and pharmacological properties of the recombinant ion/channel/receptor complex were investigated by patch clamping. Our data reveal that the cloned cDNAs code for guinea pig 5-HT3 receptors, which functionally assemble as homo-oligomers. The kinetic behavior of the ion channel and its sensitivity to several agonists and antagonists were markedly different from those of the cloned 5-HT3 receptors from mouse and human under similar experimental conditions. The agonists used were 5-hydroxytryptamine, 2-methyl-5-hydroxytryptamine, 1-phenylbiguanide (PBG), m-chlorophenylbiguanide, and the antagonists tropisetron and metoclopramide. In addition, 5-HT, PBG, and tropisetron were investigated through radioligand binding to isolated membranes. Compared with the human and murine 5-HT3 receptors, the guinea pig receptor showed prolonged desensitization kinetics. In addition, the guinea pig 5-HT3 receptor did not respond to the selective 5-HT3 receptor agonist PBG. Construction of chimeric receptors between guinea pig and human 5-HT3 receptor sequences localized the differences in desensitization kinetics to the carboxyl-terminal domain and the ligand binding site to the amino-terminal domain of the receptor protein. Molecular determinants of the PBG binding site of the human 5-HT3 receptor were localized to a 28-amino-acid spanning region adjacent to the M1 region.
Mol Pharmacol 1998 Feb
PMID:Molecular cloning, functional expression, and pharmacological characterization of 5-hydroxytryptamine3 receptor cDNA and its splice variants from guinea pig. 946 77


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