PPubMed31 Jul 2008Cited 100×

Whole proteome identification of plant candidate G-protein coupled receptors in <i> Arabidopsis </i> , rice, and poplar: computational prediction and <i> in-vivo </i> protein coupling

Timothy E Gookin, Junhyong Kim, Sarah M Assmann

Abstract

Computational prediction and in vivo protein coupling experiments identify candidate plant G-protein coupled receptors in Arabidopsis, rice and poplar.

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Computational and experimental work identified dozens of candidate plant GPCRs in Arabidopsis, rice, and poplar, with most tested proteins coupling to the G‑protein α subunit.

What’s new

i.Developed a whole‑proteome pipeline combining the QFC algorithm and three transmembrane predictors to identify candidate GPCRs in Arabidopsis, rice, and poplar.
ii.Classified the identified candidates into GPCR families and subfamilies across the three species.
iii.Experimentally validated eight high‑ranking Arabidopsis candidates using the split‑ubiquitin yeast assay, demonstrating interaction with the Gα subunit GPA1 for seven of them.
iv.Provided orthology maps and extensive supplementary datasets for the plant GPCR candidates.

How they did it

The authors applied the quasi‑periodic feature classifier (QFC) to predict GPCRs, required presence of exactly seven transmembrane domains predicted by at least two of TMHMM2, HMMTOP2, and Phobius, and filtered results with BLAST e‑value < e‑20 to find orthologs. High‑ranking candidates were then cloned and tested for interaction with the Arabidopsis Gα subunit GPA1 using the membrane‑based split‑ubiquitin yeast two‑hybrid system.

What they found

Identified 14 high‑ranking Arabidopsis candidate GPCRs, including GCR1, RGS1, and eight novel candidates (Cand1‑8).
Split‑ubiquitin assays showed 7 out of 8 tested Arabidopsis candidates (Cand1,2,3,5,7,8, HHP2) interact with GPA1, while TOM1 did not.
In Populus, 18 of 20 high‑ranking candidates were classified as class A GPCRs, with 11 belonging to the Olfactory family.
The QFC algorithm reportedly classifies GPCRs with ~98% success rate.

Caveats

✕Interaction evidence is limited to yeast split‑ubiquitin assays and requires confirmation in planta.
✕A free C‑terminal end is necessary for binding, indicating possible tagging artifacts.
✕Functional signaling (GTP exchange) was not demonstrated for the candidates.
✕Only a subset of candidates was experimentally tested, leaving many predictions unvalidated.

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Whole proteome identification of plant candidate G-protein coupled receptors in <i> Arabidopsis </i> , rice, and poplar: computational prediction and <i> in-vivo </i> protein coupling

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