Similar to all G protein subunits, the transducin G1 subunit undergoes C-terminal prenylation resulting in the addition of an isoprenoid farnesyl; however, the significance of this posttranslational modification is unclear

Similar to all G protein subunits, the transducin G1 subunit undergoes C-terminal prenylation resulting in the addition of an isoprenoid farnesyl; however, the significance of this posttranslational modification is unclear. To study the role of the farnesyl group, we genetically introduced a mutant G1 that lacked the prenylation site into the retinal photoreceptors of mice. The biochemical and physiological analyses of these mice revealed that mutant G1 dimerizes with the endogenous transducin G1 subunit and that the resulting G dimers display reduced hydrophobicity. Although mutant G dimers could form a heterotrimeric G protein, they could not mediate phototransduction. This deficiency was due to a strong exclusion of non-farnesylated G complexes from the cilia (rod outer segments). Our results provide the first evidence that farnesylation is required for trafficking of G-protein subunits to the cilium of rod photoreceptors. experiments, assessing why farnesylation of G is important Acadesine (Aicar,NSC 105823) for visual function. Some evidence suggests that posttranslational lipid modification controls transducin compartmentalization in retinal photoreceptors. For example, replacing the farnesyl group of G1 with the more hydrophobic isoprenoid group, geranylgeranyl renders the transducin G11 dimer incapable of undergoing light-driven translocation from rod outer segments (Kassai et al., 2005). In coding sequence, and a 4.4 kb mouse rhodopsin promoter (Lem et al., 1991). The cysteine-to-serine substitution at position 71, resulting in HAG1C71S mutant lacking the prenylation site, was introduced by a PCR-based strategy and the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) with the following primers: forward primer 5-AAG GAA CTC AAA GGA GGC TCT GTG ATT TCA TAG TAG G and reverse primer 5-CCT ACT ATG AAA TCA CAG AGC CTC CTT TGA GTT CCT T (with the underlined base indicating the change from wild type (WT) sequence). The integrity of both constructs was confirmed by sequence analysis. Then they were purified and injected into the pronuclei of zygotes from superovulated FVB females at the WVU Transgenic Animal Core Facility. Transgene integration was determined for both groups by PCR genotyping of tail DNA using the following primers: forward primer 5-TAC CCA TAC GAT GTT CCA GAT TAC GCT and reverse primer 5- TCA CAC Mouse monoclonal to FOXA2 AGC CTC CTT TGA GTT CCT. The colonies were established by crossing transgenic HAG1+/? and HAG1C71S+/C heterozygotes with WT partners of 129-E background (Charles River). To move both transgenes to G1-null background, HAG1+/? and HAG1C71S+/C heterozygotes Acadesine (Aicar,NSC 105823) were subjected to several round of crossing with G1?/? mice (Kolesnikov et al., 2011) to obtain HAG1+/? ; G1?/? and HAG1C71S+/C ; G1?/? mice. An identical breeding strategy using Gt1 knockout mice (Calvert et al., 2000) was used to generate HAG1+/?; Gt1?/? strain. All experiments involving mice were performed according to procedures approved by the Animal Care and Use Committee of West Virginia University. Dark Adaptation and Light Conditioning of Mice For dark adaptation, mice were kept in their original cages in the dark room for at least 12 h, and from then on, all procedures were performed under dim red light. For light conditioning, animals pupils were dilated with a mixture of 1.25% phenylephrine hydrochloride and 0.5% tropicamide ophthalmic solution for 20 min, after which mice were exposed to diffused 5000 lux white light, while free running in a white box for 10 min. Subsequently, mice were euthanized and their eyes were harvested and fixed. Quantification of Proteins by Western Blotting To quantify protein levels in isolated retinas, Acadesine (Aicar,NSC 105823) retina was dissected from the eye, gently cleaned from the contaminating tissues, and frozen on dry ice. Frozen retina was.