An Evolutionarily Conserved Arginine Is Essential for Tre1 G Protein-Coupled Receptor Function During Germ Cell Migration in Drosophila melanogaster

BACKGROUND: G protein-coupled receptors (GPCRs) play central roles in mediating cellular responses to environmental signals leading to changes in cell physiology and behaviors, including cell migration. Numerous clinical pathologies including metastasis, an invasive form of cell migration, have been linked to abnormal GPCR signaling. While the structures of some GPCRs have been defined, the in vivo roles of conserved amino acid residues and their relationships to receptor function are not fully understood. Trapped in endoderm 1 (Tre1) is an orphan receptor of the rhodopsin class that is necessary for primordial germ cell migration in Drosophila melanogaster embryos. In this study, we employ molecular genetic approaches to identify residues in Tre1 that are critical to its functions in germ cell migration. METHODOLOGY/PRINCIPAL FINDINGS: First, we show that the previously reported scattershot mutation is an allele of tre1. The scattershot allele results in an in-frame deletion of 8 amino acids at the junction of the third transmembrane domain and the second intracellular loop of Tre1 that dramatically impairs the function of this GPCR in germ cell migration. To further refine the molecular basis for this phenotype, we assayed the effects of single amino acid substitutions in transgenic animals and determined that the arginine within the evolutionarily conserved E/N/DRY motif is critical for receptor function in mediating germ cell migration within an intact developing embryo. CONCLUSIONS/SIGNIFICANCE: These structure-function studies of GPCR signaling in native contexts will inform future studies into the basic biology of this large and clinically important family of receptors

Germ cell counts showing transgenic rescue of germ cell migration in a <i>tre1^sctt</i> mutant background.

<p>The number of germ cells in the gonads of embryos in transgenic maternal rescue of the <i>tre1^sctt</i> defect was analyzed. All test constructs assayed rescue germ cell migration with the exception of the arginine to alanine substitution, <u>A</u>YILIACH. The RYILIACH construct is the positive control and the no transgene and the <i>tre1^sctt</i> reconstruction constructs are negative controls. Error bars represent the standard error of the mean (SEM).</p

Germ cell distribution in <i>tre1^sctt</i> mutants.

<p>Germ cell counts performed on stage 15–16 embryos, Mean ± S.E.M.</p>a<p>wt denotes the non-mutagenized <i>w¹¹¹⁸, P{w⁺, fat facets-lacZ}</i> parental strain.</p>b<p>two distinct phenotypic classes, presumed genotypes are based on genotyping experiments performed in Coffman <i>et al.</i> 2002.</p

Synthetic oligonucleotides used for transgenic constructs to evaluate Tre1 function.

a<p>Wild type T⁺G⁺ vector as described in Dahanukar <i>et al.</i> 2001.</p>b<p><i>tre1^sctt</i> reconstruction that lacks the eight amino acids missing in Tre1^sctt, RYILIACH.</p>c<p>Underlined sequences designate the nucleotide replacements used to create the amino acid substitutions or deletions.</p

The <i>tre1^sctt</i> mutation disrupts germ cell migration.

<p>(A-D) Dorsal views of stage 15–16 embryos are shown. Anterior is to the left. Germ cells are labeled brown with an anti-Vasa antibody. (A) In wild-type embryos, the germ cells migrate to and coalesce with the somatic gonadal precursor cells. (B) Germ cells do not migrate to the gonads in <i>tre1^sctt</i> maternal-/zygotic- (m-/z-) embryos. (C) Germ cell migration is restored in <i>tre1^sctt</i> maternal-/zygotic+ (m-/z+) embryos that have a wild-type <i>tre1</i> gene supplied paternally. (D) Germ cell migration is normal in <i>tre1^sctt</i> maternal+/zygotic- (m+/z-) embryos.</p

The arginine of the E/N/DRY motif is critical for Tre1 function in germ cell migration.

<p>Dorsal views of embryos are shown. Anterior is to the left. Stage 15–16 embryos were stained with X-Gal to visualize the <i>fat facets-lacZ</i> transgene, a germ cell marker. Embryos were from <i>tre1^sctt</i> homozygous mothers containing at least one copy of the specified transgene. The substituted amino acids are underlined. Replacement of the arginine with alanine results in a transgene that fails to rescue germ cell migration in <i>tre1^sctt</i> maternal-/zygotic- embryos. The <i>tre1^sctt</i> reconstruction lacks the 24 base pairs missing in <i>tre1^sctt</i> mutants.</p

Germ cell distribution in <i>tre1^sctt</i> maternal^-/zygotic^- embryos from mothers with modified <i>tre1</i> transgenes.

<p>Embryos were collected and aged to stages 15–16, Mean ± S.E.M.</p><p>Germ cells were detected by staining for ß-galactosidase activity using the <i>P{w⁺, fat facets-lacZ}</i> germ cell-specific marker.</p>a<p>Wild type T⁺G⁺ vector as described in Dahanukar <i>et al.</i> 2001.</p>b<p>Two independent transgenic insertions were assayed.</p