Gustducin γ subunit materials and methods
View Patent ↗Gustducin is a taste receptor cell heterotrimeric guanine nucleotide binding protein. Disclosed herein is the amino acid sequence of the gamma (γ) subunit of gustducin, as well as polynucleotide sequences encoding the γ subunit. Also disclosed are methods of modifying taste involving agents that inhibit or activate the gustducin γ subunit, methods for identifying such taste modifying agents and various taste modifying agents.
1. A method for identifying an agent which stimulates or inhibits taste comprising the steps of a) incubating gustducin γ with a G protein β subunit and phospholipase C, wherein the gustducin γ is selected from the group of (i) a polypeptide having an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 9, (ii) a polypeptide encoded by the polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8, and (iii) a polypeptide that is capable of activating phospholipase C in the presence of an agent which stimulates or inhibits taste and a G protein β subunit and that has at least 90% identity to the polypeptide of SEQ ID NO: 7; b) adding an agent; and c) measuring inositol triphosphate generation in the presence of the agent in comparison to in the absence of the agent, wherein an increase in inositol triphosphate generation in the presence of the agent indicates the agent is a taste stimulator and a decrease in inositol triphosphate generation in the presence of the agent indicates that the agent is a taste inhibitor.
2. The method according to claim 1 , wherein the G protein β subunit is Gβ1 or G62 2.
3. The method according to claim 2 , wherein the G protein β subunit is Gβ1.
4. The method according to claim 2 , wherein the G protein β subunit is Gβ2.
5. The method according to claim 1 , wherein the phospholipase C is phospholipase Cβ2.
6. The method according to claim 1 further comprising: adding 3 H-myoinositol after said incubating and prior to said measuring.
7. The method according to claim 1 , wherein the gustducin γ is a polypeptide having an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 9.
8. The method according to claim 1 , wherein the gustducin γ is a polypeptide encoded by the polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8.
9. The method according to claim 1 , wherein the gustducin γ is a polypeptide that is capable of activating phospholipase C in the presence of an agent which stimulates or inhibits taste and a G protein β subunit and that has at least 90% identity to the polypeptide of SEQ ID NO: 7.
10. The method according to claim 9 , wherein the gustducin γ is a polypeptide that has at least 95% identity to the polypeptide of SEQ ID NO: 7.