Figure 2.
Ce-grk-2 Encodes a G Protein-Coupled Receptor Kinase. (A) Predicted domain structures of Ce-GRK-2, human GRK2 (hGRK2), and human GRK3 (hGRK3) proteins. Ce-GRK-2 is 66% and 65% identical overall to hGRK3 and hGRK2, respectively. RGS, regulator of G protein signaling protein homology domain; PH, pleckstrin homology domain. The amino acid identity (%) between each human GRK and Ce-GRK-2 domain is indicated. The
rt97 mutation corresponds to a T354I change in the predicted kinase domain of Ce-GRK-2.(B) Threonine 354 is conserved among serine/threonine kinases. Predicted kinase domains were aligned using the ClustalW program (Thompson et al., 1994) in the MegAlign software package (DNASTAR). The threonine residue equivalent to T354 of Ce-GRK-2 is indicated in the gray rectangle. The signature DLG motif of GRKs (top three sequences) and the DFG motif of other kinases (bottom three sequences) are indicated by the brackets. h, human; Sc, S. ceravisiae.(C and D) A
Ce-grk-2::gfp transcriptional reporter is expressed in neurons in the body (C) and head (D). Broad expression in the nervous system and the vulval muscles is observed. Scale bar equals 100 μm in (C) and 25 μm in (D).(E and F) Ce-GRK-2 overexpressed using its own promoter can be detected in the nervous system with anti-mammalian GRK2/3 antibody. The entire animal (E) or just the head of the animal (F) are shown. Scale bar equals 100 μm in (E) and 25 μm in (F). No overt localization to sensory neuron cilia was observed, although overexpression of Ce-GRK-2 may mask or interfere with normal subcellular localization.