Prenylation-deficient G protein gamma subunits disrupt GPCR signaling in the zebrafish

Abstract

Prenylation of G protein gamma (γ) subunits is necessary for the membrane localization of heterotrimeric G proteins and for functional heterotrimeric G protein coupled receptor (GPCR) signaling. To evaluate GPCR signaling pathways during development, we injected zebrafish embryos with mRNAs encoding Gγ subunits mutated so that they can no longer be prenylated. Low-level expression of these prenylation-deficient Gγ subunits driven either ubiquitously or specifically in the primordial germ cells (PGCs) disrupts GPCR signaling and manifests as a PGC migration defect. This disruption results in a reduction of calcium accumulation in the protrusions of migrating PGCs and a failure of PGCs to directionally migrate. When co-expressed with a prenylation-deficient Gγ, 8 of the 17 wildtype Gγ isoforms individually confer the ability to restore calcium accumulation and directional migration. These results suggest that while the Gγ subunits possess the ability to interact with G Beta (β) proteins, only a subset of wildtype Gγ proteins are stable within PGCs and can interact with key signaling components necessary for PGC migration. This in vivo study highlights the functional redundancy of these signaling components and demonstrates that prenylation-deficient Gγ subunits are an effective tool to investigate the roles of GPCR signaling events during vertebrate development.

Mechanisms guiding primordial germ cell migration: strategies from different organisms

“…As in D. melanogaster, the isoprenoid branch of the pathway seems to be required, as inhibiting either Hmgcr or geranylgeranyl transferase disrupts PGC migration. Recent data suggest a role for the Hmgcr pathway in the geranylation of the Gγ subunits required directly for GPCR signalling in zebrafish PGCs 83 . Additional experiments are required to determine whether geranylation also affects the guidance of germ cells by the soma similar to D. melanogaster .…”

Gβγ signaling controls the polarization of zebrafish primordial germ cells by regulating Rac activity

“…By sequestering free G, this protein blocks the activation of its downstream effectors (Federman et al, 1992). Additionally, we expressed a mutant G2 (G2C68S), which was recently shown to interfere with the membrane localization of G and its function in PGCs (Mulligan et al, 2010). Whereas control PGCs expressing GFP were clustered in the region of the presumptive gonad at 24 hpf (Fig.…”

Identification of differentially expressed genes during development of the zebrafish pineal complex using RNA sequencing

“…It has been shown that prenylation of G protein gamma subunits is necessary to facilitate directed migration of primordial germ cells (PGC) (Mulligan et al, 2010; Mulligan and Farber, 2011). The parapineal organ also migrates directionally.…”