Mutations in the GTP-binding site of GS alpha alter stimulation of adenylyl cyclase.
Mutational replacements of specific residues in the GTP-binding pocket of the 21-kDa ras proteins (p21ras) reduce their GTPase activity. To test the possibility that the cognate regions of G protein alpha chains participate in GTP binding and hydrolysis, we compared signaling functions of normal and mutated alpha chains (termed alpha s) of Gs, the stimulatory regulator of adenylyl cyclase. alpha s chains were expressed in an alpha s-deficient S49 mouse lymphoma cell line, cyc-. alpha s in which leucine replaces glutamine 227 (corresponding to glutamine 61 of p21ras) constitutively activates adenylyl cyclase and reduces the kcat for GTP hydrolysis more than 100-fold. There is a smaller reduction in GTPase activity in another mutant in which valine replaces glycine 49 (corresponding to glycine 12 of p21ras). This mutant alpha s is a poor activator of adenylyl cyclase. Moreover, the glycine 49 protein, unlike normal alpha s, is not protected against tryptic cleavage by hydrolysis resistant GTP analogs; this finding suggests impairment of the mutant protein's ability to attain the active (GTP-bound) conformation. We conclude that alpha s residues near glutamine 227 and glycine 49 participate in binding and hydrolysis of GTP, although the GTP binding regions of alpha s and p21ras are not identical.
Masters
S B
SB
Department of Pharmacology, University of California, San Francisco 94143.
Miller
R T
RT
Chi
M H
MH
Chang
F H
FH
Beiderman
B
B
Lopez
N G
NG
Bourne
H R
HR
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras)
D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D011518:Proto-Oncogene Proteins; D006160:Guanosine Triphosphate; D004247:DNA; D000242:Cyclic AMP; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D000262:Adenylyl Cyclases; D007545:Isoproterenol