Abstract
Heterotrimeric guanine nucleotide-binding proteins are composed of alpha and beta gamma subunits and couple a variety of cell-surface receptors to intracellular enzymes or ion channels. The heterotrimer dissociates into alpha and beta gamma subunits when the alpha subunit is activated by guanine nucleoside triphosphates. Several lines of evidence show that the amino terminus of the alpha subunit is important for the interaction with the beta gamma subunit (Neer, E. J., Pulsifer, L., and Wolf, L. G. (1988) J. Biol. Chem. 263, 8996-9000; Fung, B. K.-K., and Nash, C. R. (1983) J. Biol. Chem. 258, 10503-10510). We have mutagenized the amino terminus of alpha o to dissect the relative contributions of amino-terminal myristoylation and specific amino acid sequences to subunit interaction. Wild-type and mutant alpha o cDNAs were translated in vitro in a rabbit reticulocyte lysate. All proteins were able to bind guanosine 5'-(gamma-thio)triphosphate and to achieve the necessary conformation for protection from tryptic digestion. Two assays of alpha o beta gamma interactions were used: sucrose density gradients to look for stable heterotrimer formation and ADP-ribosylation by pertussis toxin to detect weak or transient alpha o beta gamma interactions. Our results indicate that myristoylation is essential for stable heterotrimer formation, but that nonmyristoylated proteins are also capable of interacting with the beta gamma subunit. Amino acids 7-10 have an important role in alpha o beta gamma interactions whether alpha o is myristoylated or not. Deletion of this region diminishes the ability of alpha o to interact with the beta gamma subunit, but substitutions at this position indicate that other amino acids can be tolerated without affecting subunit interaction.
Highlights
As there is 141 shifts to a larger value in GDPPS. Both associated and no precedent for such an occurrence, we have made the simplest interpretation that ADP-ribosylation reflects the ability of subunits to interact with Py subunits
The rabbit reticulocyte in uitro translation system is useful for biochemical characterization of native and mutant promh * O i a r\
Acknowledgment-We thank Theresa Sladek for her expert technical assistance
Summary
Tryptic digestion of GTPyS-liganded a, pCi, >lo00 Ci/mmol, Du Pont-New England Nuclear) was added to gives a stable 37-kDa product. 1shows the in vitro translated proteins and thetryptic digestion patterns with GTPyS (added) or GTP (present in the (11%)were prepared according to Laemmli (1970). I n vitro translation of a, mRNA yields a 39-kDa product that, with trypsin and GTPyS,is converted to a 37-kDa product (Fig. 1, lanes 1 and 3 ). Ao,migrates at 37 kDa and remains at thissize after digestion with trypsin in GTPyS (Fig. 1, lanes 4-6).All the remaining mutations had the same proteolytic pattern as a. Myristoylation-Zn uitro translation was performed as described above, except that nonradioactive methionine was added to the translation mixture, and 1 pCi of [9,10-3H]myristic acid
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