Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli.
Complementary DNAs for the G protein alpha subunits Gi alpha 1, Gi alpha 2, Gi alpha 3, and Go alpha were expressed in Escherichia coli, and the four proteins were purified to homogeneity. The recombinant proteins exchange and hydrolyze guanine nucleotide, are ADP-ribosylated by pertussis toxin, and interact with beta gamma subunits. The rates of dissociation of GDP from Gi alpha 1 and Gi alpha 3 (0.03 min-1) are an order of magnitude slower than that from rGo alpha; release of GDP from Gi alpha 2 is also relatively slow (0.07 min-1). However, the values of kcat for the hydrolysis of GTP by rGo alpha and the three rGi alpha proteins are approximately the same, about 2 min-1 at 20 degrees C. The recombinant proteins restore inhibition of Ca2+ currents in pertussis toxin-treated dorsal root ganglion neurons in response to neuropeptide Y and bradykinin, indicating that the proteins can interact functionally with all necessary components of at least one signal transduction system. The two different receptors function with different arrays of G proteins to mediate their responses, since all four G proteins restored responses to bradykinin, while Gi alpha 2 was inactive with neuropeptide Y. Despite these results, high concentrations of activated Gi alpha proteins are without effect on adenylyl cyclase activity, either in the presence or absence of forskolin or Gs alpha, the G protein that activates adenylyl cyclase. These results are consistent with the hypothesis that G protein beta gamma subunits are primarily responsible for inhibition of adenylyl cyclase activity.
Linder
M E
ME
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Ewald
D A
DA
Miller
R J
RJ
Gilman
A G
AG
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000032:analysis / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005727:Ganglia, Spinal / Q000187:drug effects / Q000502:physiology; D015870:Gene Expression; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000494:pharmacology; D037342:Pertussis Toxin; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000502:physiology; D011994:Recombinant Proteins / Q000302:isolation & purification; D015398:Signal Transduction; D010566:Virulence Factors, Bordetella / Q000378:metabolism / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D015220:Calcium Channels; D009478:Neuropeptide Y; D017981:Receptors, Neurotransmitter; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D006160:Guanosine Triphosphate; D004247:DNA; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D001920:Bradykinin