Gs-activating receptors: modes of transmembrane signalling and genetic defects.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
T Gudermann
Publications and source records attributed to T Gudermann.
Human S31 serotonin receptor clone encodes a 5-hydroxytryptamine1E-like serotonin receptor.
We reported recently the molecular cloning of a genomic fragment, designated S31, that has an open reading frame of 1095 nucleotides, encoding a protein of 365 amino acids. Amino acid similarity analysis suggested that the S31 protein could be a guanine nucleotide-binding protein-coupled receptor pertaining to the serotonin receptor subfamily. Expression of the S31 open reading frame in murine L cells confirmed this, because it led to the appearance of serotonin-mediated inhibition of adenylyl cyclase activity, which was absent in the recipient L cells. We now report some aspects of the pharmacological profile of this receptor. We found that the relative potencies with which 5-hydroxytryptamine, 5-carboxamidotryptamine, methysergide, ergotamine, 8-hydroxydipropylaminotetralin, and trifluoromethylphenylpiperazine promote inhibition of adenylyl cyclase are as follows: 5-hydroxytryptamine >> methysergide >> ergotamine >> trifluoromethylphenylpiperazine > or = 8-hydroxydipropylaminotetralin > 5-carboxyamidotryptamine. This corresponds to the rank order of potencies assigned for these drugs for the 1E subtype of serotonin receptors discovered by Leonhardt and collaborators in human brain [J. Neurochem. 53:465-471 (1989)].
Adenylyl Cyclase Inhibitors↗
Molecular cloning of a truncated isoform of the human follicle stimulating hormone receptor.
Northern blot hybridization of human testicular poly (A)+ RNA to a human follicle stimulating hormone receptor probe revealed the existence of multiple mRNA transcripts. In order to investigate whether alternative splicing of the receptor occurs in the human testis we amplified the extracellular and the transmembrane domain of the human testicular follicle stimulating hormone receptor by reverse transcription polymerase chain reaction and subcloned the resulting DNA fragments. Sequence analysis of the recombinant clones revealed the existence of a truncated isoform of the human follicle stimulating hormone receptor which is spliced through a cassette exon mode without a change in the open reading frame, thereby deleting exon IX from the coding region of the receptor.
Amino Acid Sequence↗
Molecular cloning of a human serotonin receptor (S12) with a pharmacological profile resembling that of the 5-HT1D subtype.
We report the molecular cloning of a fragment of human genomic DNA called S12, containing an open reading frame of 1170 nucleotides, which encodes a receptor for serotonin of 390 amino acids. The receptor function of the S12 protein was demonstrated by functional expression in mouse LS12 cells obtained by stable transfection of Ltk- cells, and LM5S12 cells, derived from LM5 cells (Ltk- cells previously transfected with the M5 muscarinic acetylcholine receptor). Adenylyl cyclase studies showed that the S12 receptor is able to mediate inhibition of adenylyl cyclase in response to serotonin in both types of cells. As studied in LM5S12 cells, the S12 receptor did not promote Ca2+ mobilization from internal stores, nor did it significantly modulate the sustained increase in [Ca2+]i elicited by stimulation of the phospholipase C stimulating M5 acetylcholine receptor. The pharmacologic profile of S12 as seen in adenylyl cyclase assays is as follows: (EC50 in nM): serotonin, full agonist (37 nM), 5-carboxamidotryptamine, full agonist (10 nM), sumatriptan, full agonist (50 nM), metergoline, partial agonist (10 nM), methysergide, partial agonist (40 nM), yohimbine, partial agonist (150 nM), metitepin, antagonist (KB = 0.7 to 1.2 nM). We propose that the human S12 serotonin receptor is a receptor of the 5-hydroxytryptamine1D subtype.
Adenylyl Cyclase Inhibitors↗
Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells.
The murine receptor for luteinizing hormone (LHR) was cloned and expressed in L cells. This LHR (mature protein of 674 amino acids) is very similar to that of the rat (same length, 36 amino acid differences) but differs significantly more from that of man (673 amino acids, 109 differences). Expression of the murine LHR in L cells led to the appearance of binding sites for human chorionic gonadotropin (hCG) with a Kd of 150 pM and an LH- and hCG-stimulable adenylyl cyclase activity (EC50 = 50-100 pM hCG). Upon labeling pools of phosphoinositides with [3H]myo-inositol, L cells expressing the murine LHR responded to hCG with an increase in their rate of phosphoinositide hydrolysis (EC50 = 2,400 pM hCG). This was accompanied by an increase in intracellular Ca2+ [( Ca2+]i), as determined by the Fura2 method. This increase in [Ca2+]i in response to hCG was dependent on the LHR, for HCG did not affect [Ca2+]i in L cells not expressing the LHR. The effect was not due to the cAMP-forming activity of the LH receptor, for neither forskolin nor prostaglandin E1, which both increase cAMP levels in L cells, had a similar effect in either control or LHR-expressing cells and isoproterenol had no effect in L cells expressing a functionally active hamster beta-adrenergic receptor. The effect was also not due to overexpression of a Gs-coupled receptor, for L cells expressing 8-fold higher levels of the human V2 vasopressin receptor did not mimic the Ca(2+)-mobilizing response of the LH receptor. We conclude that the LH receptor has the capability of activating two intracellular signaling pathways: one leading to stimulation of adenylyl cyclase and resulting in increases in cAMP and a second leading to stimulation of phospholipase C and resulting in formation of inositol phosphates and elevations in [Ca2+]i. These data correlate positively with and provide a mechanistic explanation for previous reports on the ability of hCG to mobilize phosphoinositides and increasing [Ca2+]i in luteal and granulosa cells (e.g. Davis, J. S., West, L. A., and Farese, R. V. (1984) J. Biol. Chem. 259, 15028-15034).
Adenylyl Cyclases↗
Molecular cloning of a human gene (S31) encoding a novel serotonin receptor mediating inhibition of adenylyl cyclase.
We report the molecular cloning of human gene (S31) containing an open reading frame of 1095 nucleotides, which encodes a protein of 365 amino acids. The encoded protein contains seven hydrophobic putative transmembrane domains considered the hallmark of G protein-coupled receptors. The amino acid sequence shows highest homology to receptors for serotonin (5-hydroxytryptamine). Expression of this receptor in murine Ltk- cells conferred upon these cells the ability to respond to serotonin by inhibition of adenylyl cyclase. No response was observed to isoproterenol, epinephrine, histamine, dopamine or melatonin in the transfected cells. We propose that the human gene S31 encodes a novel serotonin receptor.
Adenylyl Cyclases↗
Report of the 7th European Workshop on Molecular and Cellular Endocrinology of the Testis.
Explore the source record for details and available documents.
Androgens↗
Ca2+ mobilization by the LH receptor expressed in Xenopus oocytes independent of 3',5'-cyclic adenosine monophosphate formation: evidence for parallel activation of two signaling pathways.
The cDNAs encoding the murine LH receptor (LHR) and the human beta 2-adrenoceptor (h beta 2AR) were cloned and RNAs complementary to their sense strands (cRNAs) were injected into defolliculated Xenopus oocytes. This led to expression, respectively, of LH- and isoproterenol-stimulable adenylyl cyclase activities, indicating that functionally active receptor cDNAs had been cloned. In oocytes injected with LHR cRNA, but not in control or h beta 2AR cRNA-injected oocytes, human CG and LH increased a Ca(2+)-activated Cl- current, as measured by the two-microelectrode voltage-clamp method. This effect was not seen with isoproterenol in control or h beta 2AR cRNA-injected oocytes, it was also not observed in response to forskolin or (Bu)2cAMP. The response to human CG could be obtained in the absence of extracellular Ca2+ but was abolished by injection of EGTA, indicating that it was caused by mobilization of Ca2+ from intracellular stores. The response was unaffected by overnight treatment with 1 microgram/ml pertussis toxin. The experiments show that a glycoprotein hormone receptor can be expressed as a functionally active molecule in Xenopus oocytes, and that the LHR has the ability of activating two separate intracellular signaling pathways: one forming the second messenger cAMP, and the other mobilizing Ca2+ from intracellular stores. It is proposed that the latter is secondary to a primary activation of phospholipase C by the LHR, which elevates intracellular Ca2+ via intermediary elevation of inositol phosphates, presumably (1,4,5)inositol trisphosphate.
Adenylyl Cyclases↗
Molecular diversity and function of G proteins and calcium channels.
General features of signal transduction by G proteins and structural properties of G-protein-modulated calcium channels are described. Recent results on roles of beta gamma dimers in signal transduction, on the kinetic properties of Gi alpha subunits and structural diversity of Go alpha subunits are discussed, as are the background and current state of our knowledge of the modulation of calcium channels by G proteins.
Amino Acid Sequence↗
The use of PCR to probe calcium channel diversity.
Voltage-dependent calcium channels are a diverse set of proteins that can be classified into at least 3 classes based on their electrophysiological and pharmacological behavior. Our studies have focused on the dihydropyridine-sensitive L-type class, which has two isoforms that have been cloned and expressed. In this report we describe the development of a polymerase chain reaction (PCR, Cetus) to probe for the expression of L-type calcium channel isoforms. We describe the optimization of the PCR reaction in terms of the following: methods for producing the template cDNA, concentration of primers, and magnesium concentration. In addition, we discuss our efforts to understand the factors involved in the design of oligonucleotides for PCR primers. These studies led to the following conclusions: 1) that primers should be less than 30 base pairs in length, 2) that the addition of extraneous polylinker sequences on the 5' end of the primer has no effect, 3) that the primer should not be located in regions where secondary structure may exist, and 4) that non-degenerate primers can be used to amplify homologous gene family members. We also present methods for subcloning PCR fragments, which allow the product of a single reaction to be subcloned and sequenced. We illustrate the use of all these techniques with RNA from mouse ovary, where we have discovered the expression of the cardiac isoform of the dihydropyridine-sensitive L-type calcium channel, and the expression of a novel sequence that we postulate to be an isoform of L-type calcium channels.
Amino Acid Sequence↗
Isolation of a protein from human ovarian follicular fluid which exerts major stimulatory effects on in vitro steroid production of testicular, ovarian, and adrenal cells.
We describe the isolation of a steroidogenesis-inducing protein (SIP) from human ovarian follicular fluid and its effects on in vitro steroid synthesis of testicular, ovarian, and adrenal cells. After heating at 60 C, precipitation with 80% ammonium sulfate and dialysis, the human ovarian follicular fluid proteins were fractionated by gel chromatography on Sephacryl S-200. The bioactivity was eluted in the mol wt region between 40 and 60 K. SIP was further purified by affinity chromatography on blue Sepharose (CL-6B), preparative isoelectrofocusing on sucrose density gradients, anion exchange chromatography on Mono Q by using fast protein liquid chromatography system and gel chromatography on Superose 12. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified SIP under both reducing and nonreducing conditions revealed a single band with an approximate mol wt of 60 K. SIP exhibited a pI value of 4.8, was heat sensitive, and lost activity upon lyophilization. SIP copurified with albumin in various isolation procedures and was distinct from human serum albumin but may represent a modified form of human albumin. SIP stimulated testosterone production by testicular pieces, interstitial cells, or purified Leydig cells from rats under different in vitro conditions. The SIP also stimulated basal and human CG stimulated in vitro progesterone production of human ovarian granulosa-lutein cells and corticosterone production of rat adrenal cells. The effects of SIP on testicular, ovarian, and adrenal cells were evident in the presence of maximal concentrations of tropic hormones. The steroid-free spent media from human granulosa-lutein cell cultures also stimulated testosterone production by rat interstitial cells, suggesting that granulosa cells may be the cellular source of SIP. In conclusion, human ovarian cells secrete a hitherto unknown albumin-like protein which enhances both basal and tropic hormone stimulated steroidogenesis in gonadal and adrenal cells. This protein may play a significant role in ovarian function by modifying steroidogenesis in the preovulatory follicle.
Adrenal Glands↗