Isoprenylation of an inhibitory G protein alpha subunit occurs only upon mutagenesis of the carboxyl terminus.
We transfected COS cells with expression vectors for the wild-type G protein alpha i1 subunit (pWT) and for mutated alpha i1 subunits, including the nonmyristylated glycine 2 to alanine mutant (pGA) and mutants in which the carboxyl termini of pWT and pGA were changed from CGLF to CVLS (pCVLS and pGA-CVLS, respectively). Immunoblot analysis of transfected COS cells with an antibody to residues 159-168 of the alpha i1 protein indicated that all four proteins were expressed. Unlike the WT and GA proteins, both CVLS mutant proteins failed to react with an antibody specific for the carboxyl terminus and failed to undergo pertussis toxin-catalyzed ADP-ribosylation. Analysis of COS cell lysates after [3H]mevalonic acid labeling indicated that specific incorporation of radioactivity occurred only in the alpha i1 subunits with the CVLS mutation. Immuno-precipitation of COS cell fractions after labeling with [35S]methionine indicated that both WT and CVLS mutant proteins were localized predominantly in the particulate fraction, whereas GA and GA-CVLS mutant proteins were found primarily in the soluble fraction. These results directly demonstrate that the carboxyl-terminal sequence, CGLF, is incapable of leading to isoprenylation but that alteration of two residues (glycine to valine, phenylalanine to serine) is sufficient to promote isoprenylation.
Jones
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TL
Molecular Pathophysiology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Spiegel
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AM
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D008715:Methionine / Q000378:metabolism; D008798:Mevalonic Acid / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D037342:Pertussis Toxin; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D009227:Myristic Acids; D011106:Polyisoprenyl Phosphates; D010566:Virulence Factors, Bordetella; D019814:Myristic Acid; D000246:Adenosine Diphosphate Ribose; D008715:Methionine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D008798:Mevalonic Acid