Identification and analysis of two splice variants of human G2A generated by alternative splicing.
G2A is a G protein-coupled receptor that can be induced by various stressors. G2A is reported to have proton-sensing activity that mediates intracellular inositol phosphate (IP) accumulation with decreasing pH. We previously showed that G2A is also activated by some oxidized free fatty acids such as 9-hydroxyoctadecadienoic acid (9-HODE). In this study, we identified a novel alternative splice variant of G2A (G2A-b) that has a partially different N terminus compared with the G2A originally reported (G2A-a). The two splice variants of G2A show similar tissue distributions, but G2A-b is expressed more abundantly. There was no difference between the two variants in 9-HODE-induced cellular responses, such as intracellular calcium mobilization and GDP/GTP exchange of Galpha protein, and in proton-sensitive IP accumulation. However, G2A-b showed a higher basal activity in terms of IP accumulation. Mutagenesis study revealed that the difference in the basal activity is attributable to the K7 residue that exists only in G2A-a. We further demonstrated that an R42A mutation largely impaired both the basal and proton-sensing activities, but did not affect the 9-HODE-induced intracellular calcium increase. Taken together, we found an additional novel G2A variant (G2A-b) that is the major transcript with functional response to ligand stimulation as well as G2A-a, and succeeded in discriminating proton-sensing and oxidized fatty acid-sensing activities of G2A.
Department of Biochemistry, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan.
Ai
A
Ogawa
Hideru
H
Obinata
Tomoyasu
T
Hattori
Mikiko
M
Kishi
Kazuaki
K
Tatei
Osamu
O
Ishikawa
Takashi
T
Izumi
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D051699:Cyclic AMP Response Element-Binding Protein A / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007962:Leukocytes; D044243:Linoleic Acids, Conjugated / Q000494:pharmacology; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D027602:Serum Response Element / Q000502:physiology; D014162:Transfection
D018797:Cell Cycle Proteins; D051699:Cyclic AMP Response Element-Binding Protein A; C115270:G2A receptor; D007295:Inositol Phosphates; D044243:Linoleic Acids, Conjugated; D020033:Protein Isoforms; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; C024347:9-hydroxy-10,12-octadecadienoic acid; D006160:Guanosine Triphosphate; D002118:Calcium