Heterotrimeric and Unconventional GTP Binding Proteins in Plant Cell Signaling

Signal-transducing GTPases in plants include small G proteins, heterotrimeric G proteins, and, potentially, several unique types of GTP binding proteins that are not members of either of the aforementioned classes. This review will focus on recent discoveries concerning the roles of heterotrimeric and “unconventional” G proteins in plant cell signaling. Small G proteins are reviewed elsewhere in this issue (Yang, 2002). Plant heterotrimeric G proteins have been the subject of several other recent reviews to which the reader is also referred (Ma, 1994; Assmann, 1996; Hooley, 1998; Bischoff et al., 1999; Fujisawa et al., 2001). Ma's review (1994) does a particularly good job of summarizing the early, mostly biochemical and immunological, progress toward identifying plant G proteins, which will not be covered here. Mammalian Heterotrimeric G-Protein Cycle. (A) Classic heterotrimeric GTPase cycle. Ligand (L) binding to a GPCR activates the associated G protein, promoting Gα and/or Gβγ interaction with downstream effectors (E). Intrinsic GTPase activity of the Gα subunit eventually returns the G protein to an inactive state. The Gα subunit remains closely associated with the plasma membrane. (B) Certain Gαs (von Zastrow and Mostov, 2001; Zheng et al., 2001) can show dissociation from the plasma membrane upon GPCR activation, contingent upon the absence or removal of lipid modification and the presence of regulatory (R) proteins. Lipid Modifiers and Effectors of G Proteins. (A) Three types of G protein lipid modifications are shown. N-Myristoylation occurs by the attachment of the saturated fatty acid myristate (blue) to a conserved acceptor Gly (red) next to the initiator Met via a stable amino bond. In palmitoylated proteins, the saturated 16-carbon fatty acid palmitate (blue) is attached to Cys residues (red) via a labile thioester bond. Protein prenylation involves the attachment of the 15- and 20-carbon isoprenes farnesyl and geranylgeranyl (blue), respectively, to conserved Cys residues (red) at the C-terminal ends of proteins via a nonreversible thioester bond. The acceptor Cys residues are part of a conserved CaaX box motif in which C indicates Cys, a represents an aliphatic amino acid, and X is usually Ser, Met, Cys, Ala, Gln, or Leu. Ct and Nt indicate C- and N-terminal amino acid positions, respectively, relative to the acceptor amino acid. Proteolysis removes the final three amino acids, and the new C-terminal Cys is then prenylated. The Arabidopsis prototypical GPA1 contains a conserved myristoylation motif and a putative sequence for palmitoylation in the N-terminal region. The two Arabidopsis G protein γ subunits (AGG1 and AGG2) contain a prenyl group binding site in the C-terminal region. (B) Cleavage sites and hydrolysis products of phospholipases regulated by G proteins. PLA2 is an acylhydrolase that specifically removes the acyl chain from the sn-2 position of the glycerol backbone (black). PLD and PI-PLC are phosphodiesterases that generate similar products, except that the phosphate group (blue) either stays with the lipid moiety (black) or goes with the head group (red). DAG, sn-1-palmitoyl,2-linoleoyl diacylglycerol; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; LysoPtdCho, sn-1-lysophosphatidylcholine; PtdCho, sn-1-palmitoyl,2-linoleoyl phosphatidylcholine; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdOH, sn-1-palmitoyl,2-linoleoyl phosphatidate. Mammals also possess ∼5 distinct Gβ and at least 12 Gγ subunits (Seack et al., 1998; Cook et al., 2001). A hallmark of the ∼35-kD Gβ proteins is the WD-40 motif, consisting of seven or eight tandem repeats and a conserved Trp-Asp (WD) motif. These repeats assemble into a seven-bladed β-propeller structure (Lambright et al., 1996; Sondek et al., 1996). Gγ proteins range in mass from 7 to 10 kD and are not highly conserved. However, all Gγs possess the C-terminal CaaX (where “a” is an aliphatic amino acid) site for isoprenylation (Figure 2A), which confers membrane association. A coiled-coil structure formed between Gβ and Gγ results in a noncovalent but very tight interaction between these two subunits, such that they function as a nondissociable dimer. A nonexhaustive list of some Gα subunit effectors in mammalian systems includes adenylate cyclase, cyclic GMP phosphodiesterase, phosphoinositide 3-kinase, the phosphoinositide (PI)-phospholipase Cβ (PI-PLCβ; Figure 2B) (Singer et al., 1997), phospholipase D (PLD; Figure 2B) (Singer et al., 1997), Na+/H+ exchange transporter (Voyno-Yasenetskaya et al., 1994; Voyno-Yasenetskaya, 1998), the TUBBY transcription factor (Santagata et al., 2001), and K+, Ca2+, and, to a lesser extent, Cl− and Na+ channels (Brown and Birnbaumer, 1990; Morris and Malbon, 1999; Reddy et al., 2001). Gβγ targets include phospholipase A2 (PLA2; Figure 2B), calcium channels, and some isoforms of adenylate cyclase and PI-PLCβ (Jelsema and Axelrod, 1987; Logothetis et al., 1987; Kim et al., 1989; Katz et al., 1992; de Waard et al., 1997; Morris and Malbon, A nonexhaustive list of some that with to mammalian heterotrimeric G protein includes members of the members of the and members of the and and this is not that in G protein are for In some G protein the from of and of are of an on Gα GTPase in Gα this is conserved all some types of Gαs by of to factor Gα on a Cys the Gα C of by with include which G proteins in which the inactive and the which activates G proteins by the of an et al., In to and such as the Arabidopsis contains Gα GPA1 et al., GPA1 is to and the conserved site for but contains the sites for and for et al., sequence and Assmann, also a palmitoylation site (Figure of GPA1 have been from several and Plant G Protein Plant G Protein GPA1 is a in but some such as have been to have two closely GPA1 et al., et al., 1996). is also a from that is to GPA1 and a of plant Gα et al., of Arabidopsis GPA1 not been GPA1 been to with a dissociation et al., The G protein been to function as a Gα by biochemical for binding for other GTPase and by et al., 1997; et al., GPA1 and protein are in all except et al., and GPA1 is particularly in and such as and and et al., et al., A similar been for et al., is highly in and et al., GPA1 been to the plasma membrane and in Arabidopsis and et al., 1997; et al., been in membrane proteins et al., A putative Gα subunit also been to the cell of the et al., 1998), et al., or channels and 1996). The of Gα by been in several not in In the acid in have acid and acid et al., and also have on in is by et al., of by not been to the does contain an sequence et al., In of which have from by also is et al., with Arabidopsis GPA1 in In of been et al., The Arabidopsis also contains Gβ with to mammalian Gβ subunits (Ma, 1994; et al., and have been in and et al., 1994; et al., 1996; et al., 1998; and et al., Arabidopsis and Gβ are in and et al., of is in in et al., 1996). closely the for with the that been to the plasma membrane as as the in with the of Gα et al., In Gβ been in plasma membrane and by protein and with with Gβ to lipid the plasma membrane et al., Lipid are of unique lipid that are to proteins, promoting association. mammalian and also to with lipid is in which are cell that are in the protein and 2001). Gγ be on the of sequence two Arabidopsis Gγ subunit and by of interaction with a Gβ protein in the and 2001). The Gγ subunits are to other and to with mammalian Gγ and possess Gγ small and a C-terminal CaaX motif, and an N-terminal with the structure to with a Gβ and also with Arabidopsis in and in binding and 2001). and are in all with an for the part to that of and 2001). G proteins in to with membrane and GTP hydrolysis by as be a G protein et al., not of a for GPA1 in recent are with such a These two of GPA1 and et al., 2001). The but show cell in and from the of the in the are of of the of the cell cycle. The of GPA1 in is with a for GPA1 in cell with this is the that that GPA1 progress the cell et al., 2001). the of Gα subunits in also cell of that and and this occurs a of cell via an G the of Gα in cell from an interaction with downstream proteins that a conserved downstream is a G protein in mammalian systems that cell in not cell types and 2001). PLA2 is by in cell and 1989; and the cell in plant and also an in to a The is in a that be in et al., et a to in the that GPA1 also a in and signaling. the Gβ a et al., 2001), that a G protein be in the of In the in similar to by the with and In with the that G in the of plant heterotrimeric G proteins have been in acid in in of the of in have from The to a G protein subunit as by several and by plants the et al., 1999; Fujisawa et al., which includes not plant as a of but also and of and et al., In the of an in by the of such as that the that the of the The as on the that not in et al., In of the and of the transcription factor to this et al., Fujisawa et al., 2001). with this that and of an and by et al., These also that Gα and the Gβ are in the A in this also a Gβ sequence with is not as et al., In to the of small or heterotrimeric G proteins et al., also in and the G protein in in the Gα subunit does not for this in can the of of as as of et al., In and at G protein also to a in the by of the downstream effectors of the is which acid and a head group from (Figure 2B) and In PLD activity in and and the of this also the of and at the of a heterotrimeric G protein in this is such a be with other that have for G protein of PLD in the of and in PLD in et al., GPA1 PLD in biochemical of PLD activity and 2001). This is is with and the that G protein the of from the membrane. PLD also is by in et al., not a interaction of Gα and PLD in this that G proteins in this cell as the by et that of is in these et al., 2001). of the channels that also is However, not all cell are of is In from the of channels that and Cl− occurs is to but is is These results that a of on in in or as a a This of by a G protein is that a mammalian channels have been to be regulated by G proteins et al., 2001). The results with the are with an of G protein of plant channels, in which that and channels of activity and Assmann, and The of by to that GPA1 to et al., have that activates in which inositol and (Figure the from and the channels and In mammalian G proteins function of the of will be to of cell is in the with this In to not and PLD but also cyclic and et al., and 2001; et al., 2001). of these by GPA1 also have the of activity by and and the by in of cell plasma membrane and Assmann, in in which the of the membrane by the not in to in this channels with the channels and are by to a of in the of the this is that activates cell channels in membrane et al., is that GPA1 to the either by interaction with the or proteins or This is by the that can channels in membrane a similar to be for the The presence of and indicate the of a of the cell to and also is some with a for G protein in et al., et al., and these have to be by a for G proteins on the is in mammalian and in to adenylate However, the of plant G protein subunits that this in plants either from via an G protein or from of the of the that the of G protein been that on to generate on the of et al., that cell can in in the is for G protein in also G proteins in in other types of plant and in and and Assmann, In of this et al., which is an to that with and will be to this that of the of is in In of the subunit as does the of However, of the a in These results the to protein in an that the et al., and de have to a G protein in a of of two channels in of which and the other of which is of the in which heterotrimeric G protein in A biochemical by and that a of GTPase activity in plasma from of The G protein is to be a protein that a GTP in a is by and is by and et al., in two G protein subunit and which of these is by remains to be similar G protein of by in a also been et al., have G proteins in and by that the of into the of by by the of of to or et al., and of binding by plant and the by et al., which activates G proteins, in and cell et al., and the of on these and et al., et al., cell and to the of heterotrimeric G proteins in the A to G protein and a of into to these in a cell and of either or with the of as downstream of the G to this is the or of the The to which this the of a cell or is not group Arabidopsis with of a either GPA1 or this to a Gα et al., 2001). that Gα to of by and but does not to is for and in a the to by GPA1 is of and GPA1 of not the GPA1 into a However, the of GPA1 in a that is not a for this These results that GPA1 at with the results of et that plants also a et from that GPA1 cell and not cell et that GPA1 by cell cell as et is that the of GPA1 upon this protein a function that does not in of of some mammalian Gαs to an of the subunit to these is to that GPA1 or GPA1 in an of Gβγ subunits, and is these Gβγ subunits that the However, this does not the that group an on cell and the other group an on cell of either cell or cell been in plant and et al., and of the is for the G proteins to be of in plant et al., and 1997; et al., 1997; 1998; et al., 1998; et al., is for the of plant heterotrimeric G proteins in to and et that stable of plants with the subunit of in to of acid, and of A of plant G proteins in plant to by the of et that an in cell the by the that the acid and the G protein activity in this to an in inositol et al., group in that from the membrane to activity and in plasma membrane also plasma membrane or these et al., by occurs with a in the and and as also from a a protein that with Gα biochemical for of a G protein et al., In group that a of GPA1 the activity of a that they is in in calcium et al., also can the of plant et that the of in of and a phospholipase and PLA2 products such as and acid in to have been to heterotrimeric G proteins in to which in is a is by of an but by also in et al., 2001), by the et al., and biochemical that and factor PLD and activity et al., 2001). G proteins also have been in the of the et al., in this as does of A plasma membrane protein from can be by conserved of mammalian Gα subunits and can plasma membrane contain a GTPase activity that is by will be to Arabidopsis the G protein and the of in that been as an in the and factor et al., and stable in calcium et al., 1997), have and as downstream of in the of in the et al., does not in et al., but the that G proteins not been been concerning the of G protein on in However, et that a heterotrimeric G protein to the is that G proteins as as et al., 1990; et al., and et al., The GPA1 subunit to the plasma membrane and the is in These sites of G protein of plant Heterotrimeric G protein subunits, and have been to the in mammalian in which in cell protein and and have been and least mammalian Gα subunit also been to the to in protein et al., include the the and the in and have been et al., Zastrow and Mostov, 2001; Zheng et al., 2001). the of prototypical in the Arabidopsis the of GPA1 and the and are have several of “unconventional” GTP binding proteins have to be group of G proteins is the G which in Arabidopsis three and and Gα putative show and to at the amino acid is similar to to been and Assmann, is the plant and a protein with a C-terminal with Gα proteins, for GTP binding as as an and a of Gαs as to small G proteins. protein GTP other the of the protein to GTPase activity and Assmann, is that some of the conserved amino from on mammalian systems to be for GTP is that the structure of the protein for the of these A unique of is N-terminal which contains a that is similar to and a The box is a sequence in proteins of the membrane in the of such as and of the presence of in the this membrane an to The box with proteins in the and this interaction is to to the membrane for in et al., the box of an The box is not in the other members of this and to have not been the plant are identifying of the to associated with and Assmann, protein in Arabidopsis with GTP binding is of the that an protein with two that are conserved in GTP binding proteins et al., are on the of and with and However, et al., that the protein is for cell in as as with cell in the of a similar to that in Arabidopsis with of GPA1 protein et al., 2001; et al., 2001). of GTP binding proteins in plants is the regulated G proteins Plant are in all at the and protein with of and protein in such as and et al., 1999; et al., The of and Arabidopsis to be regulated by the cell et al., and Arabidopsis protein been to et that a in The of this of G proteins is sequence which and 2001). is to members of this The of a and function for this of GTP binding proteins and G proteins also are Arabidopsis et al., and at the et al., in a for that small of to Gαs and to the The function of remains as a that and 1996). the from a is into a a in the for the a with sequence with protein of function et al., However, an structure for similar to that of Gα is kD and of with at the of in the and et that the protein by cell a similar to that by mammalian small G proteins as as Arabidopsis have been to in et al., 1997; and have three and three are and C are (Figure the members of a of proteins, the exchange which the exchange of GTP for on the G protein of Mammalian Heterotrimeric G Protein of Mammalian Heterotrimeric G Protein as for and the of mammalian to be a of the of this of proteins in are three as putative in of with and 1997; et al., 1998; is with the and 1997; et al., the to with the also is to have an and a C as for and of several amino acid residues as as an site that is in is at a in and et al., to be in plants a et al., 1998), but recent indicates that the by an and 2001; et al., 2001). the of remains the of mammalian show to in the which are the highly conserved of these proteins et al., 1994; and the remains that not as will be to this in of is the which possess sequence to mammalian but have been to have a et al., are to be in Arabidopsis et al., a in with the of G protein in this The is to and of a G protein is to be in this other members of the to be G protein the of GPCR in mammalian and in the recent results have that not all mammalian heterotrimeric G proteins for not all with heterotrimeric G proteins et al., 1999; In a in three mammalian proteins, to of G protein to G protein et al., 2001) is a protein to function as a GTP binding to In the proteins closely to are in the of small G proteins (Yang, 2002). is a of the protein and with Gβγ Reddy and that the Arabidopsis that have with is a protein that the dissociation of from some this with GTP binding to Gα and et al., as a dissociation a of regulatory proteins for small G proteins et al., et al., et al., the of Gα these the that with Gβγ for binding to Gαs et al., 2001), for for the of The Arabidopsis which as a of to et al., 1996; et al., 2001), is a protein with 10 repeats et al., 1996; et al., 2001) and sequence with by a G protein of the Mammalian G protein are regulated at the of the and at the of the of regulatory proteins are in is an of at the protein A and of which can be by protein and to the G protein, a as is by GPCR that which are proteins, to the et al., 1999; Morris and Malbon, 1999; 2001; and 2001). the of the of Gβγ a 2001). that have sequence with mammalian are by the Arabidopsis to and the of which to the G protein regulatory in binding to and the of the at which Gα GTP by as as and the of this to the of G protein signaling. GPCR is but the of the G protein in the presence of an This but can the exchange by to the the G protein, and the such that not but also occurs at a can of the G protein by as proteins that the G protein, and in a stable that is in the absence of et al., and in are types of mammalian proteins, proteins, and Arabidopsis PI-PLC but these of the an with but the C-terminal regulatory that is associated with activity in is particularly that the Arabidopsis not to proteins et al., 2002). proteins a of members in and 1999; Zheng et al., 1999; de et al., and and G proteins via activity and, in some via with downstream effectors for binding to Gα et al., is for plant sequence of 1998; et al., 1998; et al., 1998), on However, the of is not to the that proteins be in mammalian and have sequence at the protein an that these proteins have of that can for the that these proteins possess activity et al., new types of in plants that or sequence with mammalian can be palmitoylated and 1996). This lipid modification of the attachment of palmitate via a thioester and occurs at a conserved Cys in the C-terminal Heterotrimeric G protein and γ subunits also are subject to lipid modification (Figure of the attachment of myristate to an N-terminal Gly in Gα via the bond. of Gα palmitoylation at a Cys the and interaction with three of these Gα to the plasma membrane 1994; et al., 1999; and 2001). on the these lipid modifications also be for by regulatory proteins et al., is this lipid modification as a by which Gα and activity be and 2001; Zheng et al., 2001). in Figure GPA1 myristoylation and palmitoylation is by This to be at the biochemical in but several plant proteins contain myristoylation sites et al., 1999; et al., and have that of the myristoylation site in the protein, which in plants to et al., In palmitoylation been for the protein and These have on G protein but are they that plants possess and acyl that GPA1 as a Gγ subunits are subject to either of a or of a 20-carbon Gβ with Gγ occurs in the and prenylation occurs to this and 1996). is for with Gα and membrane association. In prenylation been as to with Gβγ for Gβγ of prenylation and 1996). in Figure and contain the conserved CaaX sequence for is by farnesyl and and or geranylgeranyl and a subunit and have distinct subunits et al., for all three of these have been in plants et al., et al., 1996; et al., 1996; et al., for have been in and et al., 1996; et al., 2001). A for the subunit from Arabidopsis et al., 2001). The subunit from et al., and an Arabidopsis to by et al., 1996). is the subunits that and have been associated with and in to in and et al., 1996; et al., The been and to and and several et al., the roles to heterotrimeric G protein in and cell be of to the prenylation and of and in the A the mammalian is the that the Arabidopsis prototypical Gα prototypical Gβ and two Gγ is that this of G proteins, of GPA1 and are not least the to these to but not to plant or proteins, which be by roles in that of The of and is the of in which GPA1 and have been and and and of the Arabidopsis is by a range of G protein of which as these be the that G proteins to Arabidopsis to that of G protein to be is that of the heterotrimeric G proteins in a the of plant and on to plant Gαs not to contain the conserved is that targets site on the plant for of the two Gα subunits contains a site and et of a protein that with Gα G elsewhere in the chain at the Gα or protein in which is not a for heterotrimeric G protein in this then the of in which heterotrimeric G proteins have been be is at to be a not in plant G protein been in G not in and but also in cell to and by of and via and/or PLD also been in several to downstream of plant heterotrimeric G proteins. via PLD is of not mammalian PLD regulatory via small heterotrimeric G proteins. the of G protein et of in is in the of the The from associated with the Gα C which is in and Arabidopsis et al., of the Arabidopsis and for the of proteins that plant heterotrimeric G proteins and will a for the to which plant G proteins have distinct roles in for the of for on also for on plant G proteins the by the and and the of of G protein in is