Gα14 and Gαq Mediate the Response to Trypsin in Xenopus Oocytes

Abstract

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 G␣ q family as possible mediators of this and other G protein-coupled receptormediated responses in Xenopus oocytes. We have cloned the third member of the G␣ q family, Xenopus G␣ 14 , in addition to the previously cloned Xenopus G␣ q and G␣ 11 (Shapira, H., Way, J., Lipinsky, D., Oron, Y., and Battey, J. F. (1994) FEBS Lett. 348, 89 -92). Amphibian G␣ 14 is 354 amino acids long and is 93% identical to its mammalian counterpart. Based on the G␣ 14 cDNA sequence, we designed a specific antisense DNA oligonucleotide (antiG␣ 14 ) that, together with antiG␣ q and antiG␣ 11 , was used in antisense depletion experiments. 24 h after injection into oocytes, either antiG␣ q or antiG␣ 14 reduced the response to 1 g/ml trypsin by 70%, whereas antiG␣ 11 had no effect. A mixture of antiG␣ q and antiG␣ 14 virtually abolished the response. These data strongly suggest that G␣ q and G␣ 14 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 G␣ q and G␣ 11 , but not G␣ 14 , to these receptors in oocytes. These results confirm the hypothesis that endogenous members of the G␣q family discriminate among different native receptors in vivo.Heterotrimeric GTP-binding proteins (G proteins) of the G␣ q family activate the phosphatidylinositol-bisphosphate-inositoltrisphosphate-calcium pathway in a pertussis toxin-insensitive manner. Among G␣ q family members, G␣ q and G␣ 11 share 88% homology and are expressed ubiquitously (2-4). G␣ 14 is 81% identical to G␣ q and is restricted in its tissue distribution mainly to spleen, lung, kidney, and testes (5). G␣ 15 and its human counterpart, G␣ 16 , show only 58 and 57% amino acid identity, respectively, to mouse G␣ q and are restricted to a subset of hematopoietic cells (5, 6). Upon activation, all G proteins of the G␣ q family activate ␤ isomers of phospholipase C.The coexistence of closely related G proteins in the same cell suggests different functions or different interactions between individual G proteins and either receptors or phospholipases. However, in reconstitution systems and transfected cells, G␣ q and G␣ 11 have been shown to couple indiscriminately to a wide range of receptors, e.g. muscarinic m1 receptor (m1-R) 1 (7, 8) and m3-R (9), thyrotropin-releasing hormone (10, 11), parathyroid hormone (12), gastrin-releasing peptide (GRP) and vasopressin (13), gonadotrophin-releasing hormone (14), angiotensin II and bradykinin (15), histamine (16), and ␣ 1 -adrenergic receptors (17). G␣ 15/16 , despite restricted distribution, are capable of coupling many serpentine receptors tested, including those natively coupled to G␣ s and G␣ i (for review, see Ref. 18). Similarly, an attempt to assign differ...

Indirect Activation of the Epithelial Na+ Channel by Trypsin

“…These receptors are classified according to their ligand specificity, with trypsin-activating PAR-2. These receptors exhibit high sensitivity to their protease substrates (32,37,50), which allows their cleavage and activation in the absence of digestion of other integral membrane proteins. It is well known that these receptors are G-protein-coupled (30 -32, 37-39).…”

“…This receptor is also endogenously expressed in oocytes and is further activated by low levels of trypsin (30,31). It is well known that this class of receptors is G-protein coupled (32,(37)(38)(39) and that the oocyte-endogenous PAR-2-mediated effects of trypsin are also coupled to a pertussis toxin-sensitive G-protein (32). To examine receptor coupling we tested: 1) the involvement of G-proteins in this response and 2) the role of endogenous PAR-2.…”

Go G‐proteins mediate rapid heterologous desensitization of G‐protein coupled receptors in Xenopus oocytes

“…The sequences of all other ASODNs were previously published (Shapira et al, 1998;Itzhaki Van-Ham et al, 2004a).…”

“…Oocytes were injected with the appropriate antisense oligodeoxynucleotides (ASODNs) (three phosphorothioate residues each at 3 0 and 5 0 ends, 50 ng/ oocyte). After injection, oocytes were maintained for additional 48-72 h in NDE96 (Shapira et al, 1998). Where desired, oocytes were injected with M1-R or thyrotropin-releasing hormone receptor (TRH-R) transcripts (2-10 ng/oocyte) to produce the expression of the appropriate receptor in 48-72 h (Lipinsky et al, 1995;Shapira et al, 1998).…”

“…Native oocytes respond to trypsin, presumably via activation of endogenous proteinase-activated receptor (PAR) (Durieux et al, 1992(Durieux et al, , 1994Schultheiss et al, 1997;Shapira et al, 1998;Ossovskaya and Bunnett, 2004). The activation of PARs involves proteolysis of the N-terminal extracellular domain of the receptor, exposing the remaining tethered peptide agonist.…”