G-protein coupled receptors (GPCRs) are a large and functionally diverse group which transmit and amplify their signals via couphng to heterotrimeric GTP binding proteins (G-proteins). Although their existence in plants has not yet been proven, an Arabidopsis EST, GCRl has been identified that has low but significant homology to a GPCR family from the cellular shme mould Dictyostelium discoideum. Apart from these Dictyostelium receptors, GCRl exhibits no significant homology to any other known genes or proteins from any organism. However, several ESTs from other plant species encode GCRl homologues, suggesting that GCRl is relatively conserved within the plant kingdom. Northern analysis of various plant tissues detected GCRl expression throughout the plant albeit at extremely low levels. Analysis of GCRl's amino acid sequence revealed the presence of seven distinct hydrophobic, membrane spanning domains, a feature common to all GPCRs. The predicted location of GCRl to a cellular membrane was investigated in vivo by fusing the green fluorescent protein (GFP) to the C-terminus of GCRl. Confocal microscopy revealed fluorescence along the outer edge of cells indicating GCRl was located on the plasma membrane. In addition to this, the predicted presence of a chloroplast transit peptide suggests that GCRl may also have a chloroplast location although this is yet to be determined experimentally.
In an attempt to suggest a function for GCRl, 83 antisense and 49 sense transgenic Lines were produced and their phenotype observed. Northern analysis of 17 of the sense lines showed that all were efficiently overexpressing GCRl and real-time quantitative RTPCR was used to prove that at least one of the antisense lines had decreased GCRl expression. No obvious phenotype was observed in any of the transgenics when grown under normal growth conditions. In addition, when subjected to the "Gauntlet" approach to screen for altered responses to plant hormones, no phenotype was observed. These results conflict with an earlier report (Plakidou-Dymock et al., 1998) which suggested GCRl may be involved in cytokinin signaling.
A yeast-two-hybrid library screen using the C-terminal tail of GCRl as bait identified a number of potential interacting proteins. One of these proteins, subsequently named PRIP, was independently isolated three times and the interaction was confirmed by an in vitro binding assay, making PRIP a strong candidate for a true interacting protein. In addition, the yeast-two-hybrid system was used to investigate the possible interaction of GCRl with heterotrimeric G-proteins. Various fragments of GCRl were tested for interaction with the Arabidopsis a, p and y subunits with negative results for every combination tested.
The evidence for GCRl as the first plant GPCR is thus limited to the smaU degree of homology to another GPCR family and the seven transmembrane domain structure. For GCRl to be labelled a genuine G-protein coupled receptor, coupling to heterotrimeric G-proteins must be demonstrated and a ligand identified.
The interacting protein, PRIP was investigated as a means to further examine GCRl's role. The full length PRIP cDNA was cloned, sequenced and homology searches indicated PRIP was a novel protein, with no significant homology to any known proteins. However, as for GCRl, several ESTs have been subsequently identified from other plant species encoding PRIP homologues, suggesting that it is conserved within the plant kingdom. PRIP was expressed throughout the green tissues of the plant with higher expression in younger tissues and no detectable expression in roots. In addition, gene expression was slightly up-regulated by wounding and down-regulated by cold-treatment. To further investigate PRIP gene expression, two kilobases of the PRIP promoter region was cloned, fused to the GUS reporter gene and introduced into Arabidopsis. Histochemical GUS assays of various tissues revealed a staining pattern that correlated with the wild-type RNA levels although no wound induction was observed. GUS staining was spread evenly across the plant with the notable exclusion of root tissues, petals and anthers. The amino acid sequence has been predicted to contain a chloroplast or mitochondrial targeting peptide and co-ordinated expression in the green tissues of the plant suggests that PRIP may be located in the chloroplast. It is possible that both GCRl and PRIP function inside the chloroplast although as GCRl is expressed in roots and has a plasma membrane location, it may serve more than one function.
The results of these characterisation studies have been unable to link GCRl to any known signaling pathways or plant processes. Nevertheless, an important groundwork has been laid for future research into the function of GCRl and its interacting partner, PRIP