G protein-coupled receptors (GPCR) constitute a large superfamily of proteins that transduce signals across cell membrane. On the exterior side, they bind to a ligand (which could be a photon, hormone, antigen, growth factor or a neurotransmitter) and at the cytosolic side, they activate a GTP binding protein (G-protein). All GPCRs share one characteristic in that they consist of a single protein chain that crosses the cell membrane SEVEN times. Loops that occur between the cell wall and the cell membrane take part in ligand recognition, while the second and third cytosolic loop and part of the C-terminal end of the receptors are implicated in G-protein recognition.
G proteins are characterized by three subunits: a, b and g. a subunit has two domains. Of the two, the function of only one, namely, the ras domain is known in somewhat detail. It contains a GDP/GTP binding site. A covalently attached lipid attaches this subunit to the lipid cell membrane bilayer. After the formation of the ligand-receptor complex, GNRP (guanine nucleotide release protein) catalyzes the removal of GDP and replaces it with GTP. Simultaneously, a subunit is dissociated from b/g-subunits. Both the GTP bound a subunit and ‘free b/g subunits can activate downstream effectors like adenyl cyclase, ion channels, Phospholipases (PL) to cause change in second messengers. The cycle turns over by the intrinsic GTPase activity of the a-subunit. It hydrolyzes GTP into GDP concomitant with reassociation of a-subunit with b/ g- subunits completing a cycle.
In the October 2002 issue of Current Opinion in Plant Biology (vol. 5: 402-407), Professor Alan M Jones at the Department of Biology, University of North Carolina, reviews the current status of research aimed at determining the novel mechanism of G-protein signaling in plant cells. After surveying the abundance of G-proteins in animals, the author calls our attention to the fact that unlike the animal kingdom, G-proteins in the model plant, Arabidopsis thaliana have only a single canonical heterotrimeric G-protein. (GPA1). GPA1 shows about 30% similarity to Gi subfamily of G-proteins in mammals.
The author’s group studied the effects of GPA1 by examining its recessive null mutant gpa1. The mutant individuals were characterized by reduced cell division; the overexpression of its wild allele resulted in extensive proliferation of the meristemic tissues. Furthermore, H.Ullah and associates of the group observed that cell division in yeast is also stimulated by the overexpression of pea G , indicating that GPA1 associates with a signal that controls cell division. Subsequent study showed that this signal could not be directly related to auxin as plants lacking G /Gb showed cell division on being treated with this hormone. Another group led by Wang et al showed that unlike auxin, a G protein couples with an abscisic acid (ABA) signaling pathway. Wang et al. demonstrated that whereas ABA treatment closes the stomata of the wild type (GPA1) under light, it has no effect on the gpa1 mutants. In these mutants there is neither ABA-induced inward K+ channels inhibition nor is there activation of pH-independent anion channels.
The author underscored the need to understand that in a specific cell type, there may exist more than one signaling mechanisms for one particular hormone such as ABA; also diverse mechanisms may operate in different cell types for the same hormone. For instance, in gpa1 mutants the guard cells are insensitive to ABA, while their seeds are as sensitive as the wild type to this hormone. The mutant seeds are markedly insensitive to gibberellic acid (GA) and completely so to brassinosteroid (BR).
One would imagine that seeds extremely sensitive to a particular hormone would not require it for germination but it cannot be said with respect to gibberellin. When GPA1 is overexpressed in seeds, they become extremely sensitive to GA and yet in its absence the seeds do not germinate. Interpreting the results, the author says, GA signaling in seed germination may not be directly linked with G-proteins. Perhaps some other G-coupled pathway may be implicated in the control of GA sensitivity. In this connection the author mentions about the BR-regulated GA sensitivity. The proof that BR regulates GA sensitivity comes from the fact that the GA-deficient seeds treated with BR germinate fully. However, Ullah et al. have shown that in instances where GA levels were reduced, BR could not restore the germination level of gpa1 seeds.
What is upstream of G in plants? In Arabidopsis, the presence of only one heptahelical transmembrane proteins structure has been confirmed in contrast to animals where the number of such units runs into the thousand or more. The transmembrane protein in Arabidopsis is called MLO1. In recessive form, it confers resistance to powdery mildew. How this gene operates is not known. Recent study shows that mildew resistance is not attributable to a G-protein coupling with this protein.
The author describes the role of G-COUPLED RECEPTOR1 (GCR1) protein that shares some sequence identity to animal GPCRs of the rhodopsin/serotonin family. The cell cycle is modified when GCR1 is overexpressed in that M phase no longer functions in synchronization with S. The author describes three proteins which he calls “Activator of G signaling1-3 [AGS1-3])”. The function of AGS is independent of a receptor similar to that reported in G-protein signaling in animals. Of the three proteins, AGS3 functions as an inhibitor to a guanine dissociation (GD1). Recently, there have been some exciting observations, namely, the carboxy-terminal domain of all plant G protein orthologs is nearly identical, while this region in animals is poorly conserved. The explanation for such a diversity is not far to seek. Unlike plants, in animals interactions are many and diverse, accounting for poor conservation of this domain. It follows that there may be a single or only a few receptors with which plant G can interact.
What is downstream of G in plants? In plants G-proteins forward signaling to effectors that regulate gene action presumably via phospholipase D (PLD), and potassium and calcium channels. Recently, it has been shown that in a G mutant, d1 affecting rice aleurone, GA-induced-amylase secretion is greatly reduced. This would be possible through the coupling of G-protein with a signal linked to GA/ABA pathway. Since PLD resides directly downstream of an activated G protein, alteration of its activity by GTPs is possible as demonstrated in aleurone membrane extracts. That G and PLD interact has also been shown in vitro. A more recent study on the gpa1 mutant suggests that GPA1 is essential for K+ influx channel activation. Heterotrimeric G protein plays another important role by activating calcium conductance in plant cells. G proteins have also been shown to increase the probability of channel opening.
Conclusions: G-coupling in Arabidopsis differs markedly from that which operates in animals. In this model plant, only one or two G-protein complexes are implicated in coupling a particular signal to a particular effect. The author introduces a concept that there may be a selective confinement of receptors and effectors with G to discrete micro-domains called “rafts”. In such structures, which have been reported in plants, all the components coupled with G may be physically held together. Whether a heterotrimeric G-protein is a component of such rafts is not known.
From what is known so far, it seems that a number of signals which were considered to lie on a multitude of pathways may only be indirectly regulated by G proteins. The author ends on a confident note that with new genetic tools now available along with an essentially complete Arabidopsis genome in hand, it may be possible to resolve the complexity concerning G-protein-coupled signaling in plants in general and Arabidopsis in particular.
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