Gustducin materials and methods

Abstract

A novel taste cell specific guanine nucleotide binding protein, gustducin, is disclosed as well as polynucleotide sequences encoding the α subunit of gustducin. Also disclosed are methods of modifying taste involving agents that inhibit or activate the gustducin α subunit, methods for identifying such taste modifying agents and various taste modifying agents.

1 . A purified and isolated polynucleotide sequence encoding the α subunit of gustducin or a fragment or a variant thereof, wherein the encoded gustducin product possesses at least one ligand/antiligand binding activity or immunological property specific to gustducin.  
     
     
         2 . The polynucleotide sequence of  claim 1  which is a CDNA sequence or a biological replica thereof.  
     
     
         3 . The polynucleotide sequence of  claim 2  wherein said sequence is SEQUENCE ID NO: 20.  
     
     
         4 . The polynucleotide sequence of  claim 1  which is a genomic DNA sequence or a biological replica thereof.  
     
     
         5 . The polynucleotide sequence of  claim 1  which is a chemically synthesized DNA sequence or a biological replica thereof.  
     
     
         6 . The polynucleotide sequence of  claim 1  wherein said sequence encodes the α subunit of human gustducin.  
     
     
         7 . A biologically functional DNA vector comprising the polynucleotide sequence of  claim 1 .  
     
     
         8 . A host cell stably transformed or transfected with the polynucleotide sequence of  claim 1  in a manner allowing the expression in said host cell of a gustducin α subunit polypeptide, fragment or variant, wherein the gustducin expression product possesses at least one ligand/antiligand binding activity or immunological property specific to gustducin.  
     
     
         9 . A method for producing a gustducin α subunit polypeptide, fragment or variant wherein the gustducin product possesses at least one ligand/antiligand binding activity or immunological property specific to gustducin, comprising the steps of growing a host cell stably transformed or transfected with the polynucleotide sequence of  claim 1  in suitable nutrient medium and isolating the gustducin product from said cell or from the medium of its growth.  
     
     
         10 . Purified and isolated gustducin α subunit polypeptide, fragment or variant possessing at least one ligand/antiligand binding activity or immunological property specific to gustducin.  
     
     
         11 . An antibody substance specific for the α subunit of gustducin.  
     
     
         12 . A method for identifying a taste modifying agent comprising the steps of incubating phospholipid vesicles having gustducin α subunit or transducin α subunit and G protein β and γ subunits associated in biologically active form with said agent and GTPγS, and determining the rate of GTPγS binding by said α subunit in comparision to a standard rate wherein an increase in the rate of binding indicates that the agent is a taste stimulator and a decrease in the rate of binding indicates that the agent is a taste inhibitor.  
     
     
         13 . A method for identifying a taste modifying agent comprising the steps of incubating phospholipid vesicles having gustducin α subunit or transducin α subunit and G protein β and γ subunits associated in biologically active form with said agent and GTP, and determining the rate of conversion of GTP to GDP by said α subunit in comparison to a standard rate wherein an increase in the rate of conversion indicates that the agent is a taste stimulator and a decrease in the rate of conversion indicates that the agent is a taste inhibitor.  
     
     
         14 . A method for identifying a taste modifying agent comprising the steps of incubating activated gusducin α subunit or transducin α subunit with said agent and a phosphodiesterase, and measuring phosphodiesterase activation by said α subunit in comparison to a standard wherein an increase in phosphodiesterase activity indicates that the agent is a taste stimulator and a decrease in phosphodiesterase activity indicates that the agent is a taste inhibitor.  
     
     
         15 . A method for identifying a taste modifying agent comprising the steps of: 
 (a) incubating, washed disk membranes having gustducin α subunit or transducin α subunit associated with G protein β and γ subunits in biologically active form with said agent;    (b) subjecting said membranes to photolyzing conditions; and    (c) determining absorption at 380 nm in comparison to a standard wherein an increase in absorption at 380 nm indicates the agent is a taste stimulator and a decrease in absorption at 380 nm indicates that the agent is a taste inhibitor.    
     
     
         16 . A taste modifying agent comprising a peptide possessing at least one ligand/antiligand binding activity of the α subunit of gustducin.  
     
     
         17 . A taste modifying peptide wherein said peptide has an amino acid sequence selected from the group consisting of: 
 (a) SEQ ID NO: 1;    (b) SEQ ID NO: 2;    (c) SEQ ID NO: 3;    (d) SEQ ID NO: 11;    (e) SEQ ID NO: 12; and    (f) SEQ ID NO: 13.    
     
     
         18 . A method for identifying a peptide ligand/antiligand of gustducin comprising the steps of contacting gustducin α subunits with peptides and isolating peptides which bind to said subunits.  
     
     
         19 . A method for modifying taste comprising the step of delivering to taste receptor cells a taste modifying agent selected from the group consisting of: 
 (a) the taste modifying agent of claim  16 ;    (b) the taste modifying peptide of SEQ ID NO: 1;    (c) the taste modifying peptide of SEQ ID NO: 2;    (d) the taste modifying peptide of SEQ ID NO: 3;    (e) the taste modifying peptide of SEQ ID NO: 4;    (f) the taste modifying peptide of SEQ ID NO: 5;    (g) the taste modifying peptide of SEQ ID NO: 6;    (h) the taste modifying peptide of SEQ ID NO: 7;    (i) the taste modifying peptide of SEQ ID NO: 8;    (j) the taste modifying peptide of SEQ ID NO: 9;    (k) the taste modifying peptide of SEQ ID NO: 10;    (l) the taste modifying peptide of SEQ ID NO: 11;    (m) the taste modifying peptide of SEQ ID NO: 12;    (n) the taste modifying peptide of SEQ ID NO: 13;    (o) the taste modifying peptide of SEQ ID NO: 14;    (p) the taste modifying peptide of SEQ ID NO: 15;    (q) the taste modifying peptide of SEQ ID NO: 16;    (r) the taste modifying peptide of SEQ ID NO: 17;    (s) the taste modifying peptide of SEQ ID NO: 18; and    (t) the taste modifying peptide of SEQ ID NO: 19.    
     
     
         20 . A method for modifying taste comprising the step of delivering the antibody substance of  claim 11  to taste receptor cells.