Abstract
G protein-mediated signal transduction is dependent on alpha subunit interactions with beta gamma subunits, receptors, effectors, magnesium ions, and guanine nucleotides. The interdependence of these interactions can be probed by mutational analysis. We developed large scale screening procedures in recombinant Escherichia coli to identify and characterize novel mutations in G(o) alpha. Random mutations were generated by polymerase chain reaction in the amino-terminal 56 amino acids of G(o) alpha. Guanine nucleotide binding properties of the mutants were assayed in situ and in crude extracts of recombinant E. coli. beta gamma interactions were assayed by pertussis toxin mediated ADP-ribosylation. Efficacy of the screening procedures was evaluated by studying properties of wild-type G(o) alpha and site-directed mutations that were characterized previously in other G proteins. Several novel mutants with altered GTP binding characteristics and reduced ability to interact with beta gamma had been isolated from the randomly generated mutant library. ADP-ribosylation of mutants R10G, K21N, and K35E was significantly reduced, whereas two of the mutants bearing multiple amino acid substitutions were refractory to modification. Mutant K35E also exhibited reduced affinity to guanosine 5'-(3-O-thio)triphosphate at submicromolar concentrations of magnesium. These experiments demonstrate the feasibility of using large scale random mutagenesis in the studies of G protein function.
Highlights
Gprotein-mediatedsignal transduction is dependent on a subunit interactions with By subunits, receptors, effectors, magnesium ions, and guanine nucleotides
Site-directed mutagenesis has been basedmostly upon analogy with the extensive mutagenesis of Ras [13], focusing primarily on the conserved amino acid regions that areinvolved in Ga subunit interactionwith guanine nucleotides [14,15,16,17,18] or studies of chimeric G. and Gi the screening procedures was evaluated by studying a subunits [19, 20]
In agreement with the previous ribose into the mutant K35E was less than 5% of the wild- studies of other G proteins, the Q205L mutation in Goa type recombinant Goa protein (rGoa), even at a 5-fold excess of 0-y over the a subunit. inhibited GTPase activity, (Figs. 4 and 5), whereas G203T, The extract fromcells expressing the 4Gll rGoa had no G204A, and the double mutant GG203,204TAaffected the inhibitory effect on the labeling of the wild-type Goa, i.e. its nucleotide binding (Fig. 3)
Summary
From the Biology Division, CaliforniaZnstitute of Technology, Pasadena, California 91125. Goa.Random mutationswere generated by polymerase chain reaction in the amino-terminal 56 amino acids of Goa. Guanine nucleotide binding properties of the mutants were assayed in situ and in crude extracts of recombinant E. coli. Teins we intend to develop a high resolution missense mutagenesis map of the a subunit thatwill cover the entire protein sequence To reach this goal, it will be necessary to generate a large number of random mutations in different partosf the a subunit protein, express the mutatedcDNA in Escherichia coli, and characterize recombinant mutant protein in crude extracts from isolatecdlones. Screening of the Mutant Library for GTP-yS Binding-For induction of Goaand N-myristoyltransferase expression, bacterial colonies were lifted to nitrocellulose filters, transferred to agar plates containing 0.5 mM IPTG, and incubated at room temperature for 3 h.
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