Abstract
The baculovirus-based expression system was adapted to express alpha subunits of the complete (alpha i3) and an amino-terminally truncated (alpha i3') form of Gi3 and of two complete forms of Gs (alpha s-L and alpha s-S). Subunits encoded in full length cDNAs were obtained with yields of 40-60 mg of recombinant protein/liter of cells, of which alpha i3 was between 30 and 50% soluble, but alpha s subunits were only 5-10% soluble. Only the complete alpha i3 was myristoylated. alpha i3 was purified in four steps. The purified protein bound 0.8-0.9 mol of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) per mol of protein and had one predominant contaminant which was identified as a truncated form that begins with methionine 18 instead of methionine 1. Both the full length alpha i3 and the truncated alpha i3' formed trimers with human erythrocyte beta gamma as seen by their migration in sucrose density gradients and by an increased rate of ADP ribosylation by pertussis toxin, but compared to alpha i3, alpha i3' interacted with beta gamma with a reduced affinity and dissociated upon warming. At 32 degrees C, only full length alpha i3 was ADP-ribosylated; at 4 degrees C, alpha i3 and alpha i3' were both ADP-ribosylated, with the truncated form requiring approximately 200-fold higher concentrations of beta gamma. A genetically engineered alpha i3' (alpha i3[18-354]) was also expressed in Sf9 cells. Yields, assessed as saturable GTP gamma S binding sites, were 3-5 mg per liter. Scatchard analysis showed that truncation of the amino terminus interferes with the ability of Mg2+ to promote high affinity binding of GTP gamma S. We conclude that the G protein alpha subunit amino terminus is not essential for interaction with beta gamma dimers, but rather is important in determining the affinity of the alpha subunit for both the beta gamma dimers and guanine nucleotide.
Highlights
We have demonstrated that theremoval of a defined piece terminalamino acid of the trypsin-induced truncations is of the amino terminuosf an a subunit reduces its affinityfor Asn2*,four positions downstream from the Met'8 that constitutes the amino terminus of ourtruncated form of the a The “catalytic”property of Py as seen by an EC, to subunit
At4 “C the apy complex may by an alternate approach involving site directed mutation of be less stable, and a dissociation might not lead to a deactithese four amino acids in the context of a n otherwise unal- vation of By
Thicso, mbined with the fact that the truncated form has as its initial amino acid Met”, suggests that its mosltikely origin is a secondary initiationat
Summary
The a subunits of two forms of G, (a,-L and as-S;both without Ser) and of Gi3 (ai3)w, ere subcloned and expressed following modifications of the procedures of Summers and colleagues described under "Experimental Procedures." The proteinstainingpattern of total cell lysates of Sf9 cells infected with recombinant baculovirus indicated that full length a subunits were expressed at high levels, constituting as much as 10-15% of the total protein of these cells (not shown). Myristoylution-It has been shown in several instances that some a subunits are post-translationally modified by adding a myristoyl group at the amino terminus We tested this in cell culture by infection with recombinant virus and 3 days later by addition of radiolabeled myristic acid or palmitic acid. Upon longer exposure of the film, incorporation of low levels of radiolabeled palmitic acidwas detected in all three recombinant products, while infected controls (recombinant VP6 or wild type polyhedrin) showed no visible incorporation (not shown) From this we conclude that the cells properly modify the amino terminus of a i 3 by first clipping the methionine with an aminopeptidase, adding the myristic acid to the glycine residue as the acceptor site. TheEC60with which M e reduced GTPyS dissociation was 40-60 nM in reasonable agreement with previous studies indicating thepresence of a high affinity binding site for M e on human erythrocyteGi3protein
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