Galpha14 and Galphaq mediate the response to trypsin in Xenopus oocytes.
Xenopus oocytes respond to trypsin with a characteristic chloride current, virtually indistinguishable from responses mediated by a large number of native and expressed G protein-coupled receptors. We studied the involvement of G proteins of the Galphaq family as possible mediators of this and other G protein-coupled receptor-mediated responses in Xenopus oocytes. We have cloned the third member of the Galphaq family, Xenopus Galpha14, in addition to the previously cloned Xenopus Galphaq and Galpha11 (Shapira, H., Way, J., Lipinsky, D., Oron, Y., and Battey, J. F. (1994) FEBS Lett. 348, 89-92). Amphibian Galpha14 is 354 amino acids long and is 93% identical to its mammalian counterpart. Based on the Galpha14 cDNA sequence, we designed a specific antisense DNA oligonucleotide (antiGalpha14) that, together with antiGalphaq and antiGalpha11, was used in antisense depletion experiments. 24 h after injection into oocytes, either antiGalphaq or antiGalpha14 reduced the response to 1 microg/ml trypsin by 70%, whereas antiGalpha11 had no effect. A mixture of antiGalphaq and antiGalpha14 virtually abolished the response. These data strongly suggest that Galphaq and Galpha14 are the exclusive mediators of the trypsin-evoked response in Xenopus oocytes. Similar experiments with the expressed gastrin-releasing peptide receptor and muscarinic m1 receptor revealed the coupling of Galphaq and Galpha11, but not Galpha14, to these receptors in oocytes. These results confirm the hypothesis that endogenous members of the Galphaq family discriminate among different native receptors in vivo.
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel. hshapira@post.tau.ac.il
H
H
Shapira
I
I
Amit
M
M
Revach
Y
Y
Oron
J F
JF
Battey
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000737:chemistry*; D007700:Kinetics; D008845:Microinjections; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology*; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000378:metabolism; D018004:Receptors, Bombesin / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D014357:Trypsin / Q000494:pharmacology*; D014981:Xenopus
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D018004:Receptors, Bombesin; D011976:Receptors, Muscarinic; D014357:Trypsin; D019204:GTP-Binding Proteins