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. 2011 Oct;23(10):1617-24.
doi: 10.1016/j.cellsig.2011.05.015. Epub 2011 Jun 15.

Central and C-terminal domains of heterotrimeric G protein gamma subunits differentially influence the signaling necessary for primordial germ cell migration

Timothy Mulligan  1 , Steven A Farber
Affiliations

Central and C-terminal domains of heterotrimeric G protein gamma subunits differentially influence the signaling necessary for primordial germ cell migration

Timothy Mulligan et al. Cell Signal. 2011 Oct.

Abstract

Heterotrimeric G protein signaling is involved in many pathways essential to development including those controlling cell migration, proliferation, differentiation and apoptosis. One key developmental event known to rely on proper heterotrimeric G protein signaling is primordial germ cell (PGC) migration. We previously developed an in vivo PGC migration assay that identified differences in the signaling capacity of G protein gamma subunits. In this study we developed Gγ subunit chimeras to determine the regions of Gγ isoforms that are responsible for these differences. The central section of the Gγ subunit was found to be necessary for the ability of a Gγ subunit to mediate signaling involved in PGC migration. Residues found in the carboxy-terminal segment of Gγ transducin (gngt1) were found to be responsible for the ability of this subunit to disrupt PGC migration. The type of prenylation did not affect the ability of a Gγ subunit to reverse prenylation-deficient-Gγ-induced PGC migration defects. However, a version of gng2, engineered to be farnesylated instead of geranylgeranylated, still lacks the ability to reverse PGC migration defects known to result from treatment of zebrafish with geranylgeranyl transferase inhibitors (GGTI), supporting the notion that Gγ subunits are one of several protein targets that need to be geranylgeranylated to orchestrate the proper long-range migration of PGCs.

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Figures

Fig. 1
Fig. 1
Strategy for constructing Gγ subunit chimeras. The gng2 and gng15 (A) or gng2 and gngt1 (B) subunits were aligned with Clustal to show their similarities and differences at the amino acid level. Gγ subunits were split at the conserved sites boxed in red (QLK which corresponds to residues 18–20 in gng2 and DPL, which corresponds to residues 48050 of gng2). The residues that make up the CaaX motif (blue box), and the N-terminal, middle and C-terminal sections have been indicated. The three sections of each gamma subunit were individually PCR amplified and recombined to form the chimeras outlined in (C). Chimeras were named according to the makeup of their three sections as indicated to the left of each subunit Swapping the ‘translocation to endomembranes’ motif (*) involved mutating the five amino acids indicated in an alignment of their C-terminal sequences (D). Subunits harboring these point mutations were named according to whether their new motif should (trans+) or should not (trans-) be consistent with the ability to translocate upon receptor activation. Subunits with swapped CaaX motifs have been named with the amino acids that make up their new motif (E) (gng2-WT terminates with -CAIL; after swap = gng2-CVIC). The prenyl lipid that will be post-translationally added to these Gγ chimeras is indicated to the right.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Ability of the Gγ2/Gγ15 subunit chimeras to reverse gng2-SaaX-induced PGC migration defects. Embryos were injected with 50 pg of gng2-SaaX(nos) mRNA (scored when injected alone on the leftmost in red) and received a subsequent injection with 100, 200, or 300 pg of the gng15/gng2 chimeric Gγ mRNA (A). The ectopic PGC score of larvae injected with 100–300 pg/embryo of the chimeric Gγ-WT(nos) mRNA alone is shown in (B). Each concentration is summarized from 2 to 5 experiments with a minimum of 20 larvae scored for the number of ectopic PGCs at 24 hpf. (Ectopic PGC migration score is expressed as mean +/− S.D.) An ectopic score of 0 represents wild type migration; 1=5–20% ectopic; 2=21–40% ectopic; 3=41–60% ectopic; 4=61– 80% ectopic; 5=81–100% ectopic PGCs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
Ability of the Gγ2/Gγt1 subunit chimeras to reverse the gng2-SaaX-induced PGC migration defects. Embryos were injected with 50 pg of gng2-SaaX(nos) mRNA (scored when injected alone on the leftmost in red) and received a subsequent injection with 100, 200, or 300 pg of the gngt1/gng2 chimeric Gγ mRNA (A). The ectopic PGC score of larvae injected with 100–300 pg/embryo of the chimeric Gγ-WT(nos) mRNA alone is shown in (B). Each concentration is summarized from 2 to 5 experiments with a minimum of 20 larvae scored for the number of ectopic PGCs at 24 hpf. (Ectopic PGC migration score is expressed as mean +/− S.D.) An ectopic score of 0 represents wildtype migration and a score of 5=81–100% ectopic PGCs. The amino acids swapped in the Gγ translocation motif chimeras (*) are listed in Figs. 4–1. Wildtype Gγ subunits and chimeras with swapped CaaX motifs have been indicated with whether they are to be post translationally modified with the 15-carbon farnesyl or 20-carbon geranylgeranyl lipid. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Ability of Gγ subunit chimeras to reverse the PGC migration defects caused by GGTI-2166. (GGTI-2166 injection) Embryos were injected with 2 nL of a mixture of 2.5 mM GGTI-2166 and 150 pg GFP-CVLL(nos). Injected embryos were split into three groups and received a 1 nL injection with either 1.) phenol red (injection control), 2.) 200 pg gng2-WT(nos) mRNA or 3.) 200 pg gng2-CVIC(nos) mRNA. PGC migration was assayed at 24 hpf. (GGTI-2166 soaking) Embryos were injected with 150 pg GFP-CVLL (nos) mRNA either alone or in a mixture with 200 pg gng2-WT(nos) mRNA or 200 pg gng2-CVIC(nos) mRNA. Injected embryos were soaked for 24 h in 2% ethanol (control) or 20 μM GGTI-2166 (final ethanol concentration=2%). PGC migration was assayed at the end of the incubation period. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5
Alignment of the Gγ subunit regions responsible for differences in signaling capabilities. The C-terminal region of gngt1, which was found to confer upon the gamma subunit the ability to disrupt PGC migration when injected in its native-CaaX form, is aligned to the C-terminus of gng2 (A). The central region of the gamma subunit confers a difference in ability to reverse gng2-SaaX-induced defects. Clustal alignment of this region highlights the sequence differences among the gngt1, gng2 and gng15 subunits (B). Shown to the right of each of the cartoon schematics of the Gγ subunit chimeras is their ability to disrupt PGC migration (A) or reverse the gng2-SaaX-induced defects (B).
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References

    1. Wettschureck N, Offermanns S. Physiol Rev. 2005;85:1159–1204. - PubMed
    1. Hurowitz EH, Melnyk JM, Chen YJ, Kouros-Mehr H, Simon MI, Shizuya H. DNA Res. 2000;7:111–120. - PubMed
    1. Mulligan T, Blaser H, Raz E, Farber SA. Cell Signal. 2010;22:221–233. - PMC - PubMed
    1. Oka Y, Saraiva LR, Kwan YY, Korsching SI. Proc Natl Acad Sci U S A. 2009;106:1484–1489. - PMC - PubMed
    1. Iniguez-Lluhi JA, Simon MI, Robishaw JD, Gilman AG. J Biol Chem. 1992;267:23409–23417. - PubMed

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