title: Differential coupling of G protein alpha subunits to seven-helix receptors expressed in Xenopus oocytes.
abstract: Xenopus oocytes were used to examine the coupling of the serotonin 1c (5HT1c) and thyrotropin-releasing hormone (TRH) receptors to both endogenous and heterologously expressed G protein alpha subunits. Expression of either G protein-coupled receptor resulted in agonist-induced, Ca(2+)-activated Cl- currents that were measured using a two-electrode voltage clamp. 5HT-induced Cl- currents were reduced 80% by incubating the injected oocytes with pertussis toxin (PTX) and inhibited 50-65% by injection of antisense oligonucleotides to the PTX-sensitive Go alpha subunit. TRH-induced Cl- currents were reduced only 20% by PTX treatment but were inhibited 60% by injection of antisense oligonucleotides to the PTX-insensitive Gq alpha subunit. Injection of antisense oligonucleotides to a novel Xenopus phospholipase C-beta inhibited the 5HT1c (and Go)-induced Cl- current with little effect on the TRH (and Gq)-induced current. These results suggest that receptor-activated Go and Gq interact with different effectors, most likely different isoforms of phospholipase C-beta. Co-expression of each receptor with seven different mammalian G protein alpha subunit cRNAs (Goa, Gob, Gq, G11, Gs, Golf, and Gt) was also examined. Co-expression of either receptor with the first four of these G alpha subunits resulted in a maximum 4-6-fold increase in Cl- currents; the increase depended on the amount of G alpha subunit cRNA injected. This increase was blocked by PTX for G alpha oa and G alpha ob co-expression but not for G alpha q or G alpha 11 co-expression. Co-expression of either receptor with Gs, Golf, or Gt had no effect on Ca(2+)-activated Cl- currents; furthermore, co-expression with Gs or Golf also failed to reveal 5HT- or TRH-induced changes in adenylyl cyclase as assessed by activation of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results indicate that in oocytes, the 5HT1c and TRH receptors do the following: 1) preferentially couple to PTX-sensitive (Go) and PTX-insensitive (Gq) G proteins and that these G proteins act on different effectors, 2) couple within the same cell type to several different heterologously expressed G protein alpha subunits to activate the oocyte's endogenous Cl- current, and 3) fail to couple to G protein alpha subunits that activate cAMP or phosphodiesterase.
authors.affiliation: Division of Biology, California Institute of Technology, Pasadena 91125.
authors.forename: M W
authors.initials: MW
authors.lastname: Quick
authors.forename: M I
authors.initials: MI
authors.lastname: Simon
authors.forename: N
authors.initials: N
authors.lastname: Davidson
authors.forename: H A
authors.initials: HA
authors.lastname: Lester
authors.forename: A M
authors.initials: AM
authors.lastname: Aragay
mesh_terms: D037361:Adenylate Cyclase Toxin; D000818:Animals; D001483:Base Sequence; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D009865:Oocytes; D037342:Pertussis Toxin; D018075:RNA, Complementary / Q000378:metabolism; D011985:Receptors, Serotonin / Q000096:biosynthesis / Q000378:metabolism*; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000096:biosynthesis / Q000378:metabolism*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014981:Xenopus
chemical_list: D037361:Adenylate Cyclase Toxin; D018118:Chloride Channels; D007527:Isoenzymes; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D018075:RNA, Complementary; D011985:Receptors, Serotonin; D018025:Receptors, Thyrotropin-Releasing Hormone; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins