Journal of Molecular Biology
Regular articleSolution structure of human GAIP (Gα interacting protein): a regulator of G protein signaling1
Introduction
Many of the processes necessary for cell life depend on signaling pathways through which the environments inside and outside the cell are connected. The information from the cell exterior is processed by means of a cascade of molecular interactions until the desired response is achieved. This is the so-called signaling pathway. There are proteins responsible for switching it on and off, that is, making the cell able or not to respond to external signals. One of the most important protein families having this function are G proteins Freissmuth et al 1989, Kaziro et al 1991, Hepler and Gilman 1992. These are GTP (guanosine triphosphate) binding proteins and are heterotrimers consisting of subunits α, β and γ. When G proteins are inactive (the signaling pathway is switched off), the α-subunit is bound to the β and γ-subunits and to GDP (guanosine diphosphate). In contrast, when the G protein is active and the signaling pathway is on, the α-subunit is dissociated from the β-γ complex and is bound to GTP. The inactivation of the pathway is accomplished through GTP hydrolysis to GDP, therefore, the rate of GTP hydrolysis determines the time the G protein is active and consequently the duration of the physiological response.
Although G proteins are GTPases, there are other proteins that help them to perform this function. This type of proteins are called GTPase-activating proteins or GAPs. Recently, a novel family of regulators of G protein signaling (RGS) has been identified De Vries et al 1995, Druey et al 1996, Koelle and Horvitz 1996, De Vries and Farquhar 1999. All of its biochemically studied members have been shown to act as GAPs (Berman & Gilman, 1998). RGS proteins share a ∼130 residue core, called the RGS box, that accounts for the GAP activity Faurobert and Hurley 1997, Popov et al 1997. Nevertheless, the emerging biochemical information concerning the RGS protein family suggests that a more complex scenario is taking place. For example, it has been shown that the GAP function can be dependent on the type of receptor that interacts with the G protein (Xu et al., 1999). There exists evidence that full-length RGS proteins are much more efficient as GAPs than the RGS box alone when tested in vivo (Chen & Lin, 1998) and it has been suggested that the regulation by RGS proteins could be related not only to their GAP activity but also to a competition for effector binding (Hepler et al., 1997).
Another important characteristic of RGS proteins is that most of them seem to interact preferentially with the Gαi class De Vries et al 1995, De Vries et al 1998a, Berman et al 1996a, which includes Gαi1, Gαi2, Gαi3, Gαo, Gαt and Gαz. The Gαi subfamily is involved in functions such as the inhibition of adenylyl cyclases, the activation of K+ and Ca2+ channels and the activation of cyclic guanosine monophosphate phosphodiesterases (Neer, 1995). Interaction and GAP activity toward members of the Gαq and Gα12 (regulators of K+ and Na+ channels) subfamilies have been evidenced Kozasa et al 1998, Hart et al 1998, but apparently RGS do not show affinity for the Gαs (stimulators of adenylyl cyclases) class. From the different RGS proteins the most widely studied in biochemical terms are RGS4 (Druey et al., 1996) and GAIP De Vries et al 1995, De Vries et al 1996. Concerning the RGS box, there is not a clear distinction between the specificity of the two, although it has been shown that GAIP is a slightly more efficient GAP for Gαz than RGS4 Popov et al 1997, Wang et al 1998, while the opposite occurs for Gαq (Hepler et al., 1997). In addition, it has been suggested that GAIP, RGSZ1 and RET-RGS1 form a subfamily of Gz GAPs within the RGS proteins (Wang et al., 1998). With respect to parts of the sequence outside the RGS box, GAIP and RGS4 have different functions and possibly different localizations De Vries et al 1998a, De Vries et al 1998b.
The interaction between RGS and Gα is favored when the latter is either bound to GTP or bound to GDP and AlF4− (Berman et al., 1996b). This last form has been shown to be an analog of the transition state of GTP hydrolysis (Coleman et al., 1994).
We have determined the solution structure of the human regulator GAIP by NMR in order to give insight into the structure-function relationships of this protein family.
Section snippets
GAIP structure determination
Although GAIP is a 217 residue protein, we have studied only the 128 residue core known to be responsible for its Gα interaction (De Vries et al., 1995) and GAP activity Popov et al 1997, Fischer et al 1999. GAIP backbone and side-chain resonance assignments have been obtained using 15N and 15N,13C-labeled samples for heteronuclear magnetic resonance experiments (Materials and Methods). Only five amide resonances have not been observed in the NMR spectra, probably due to their exchange
Conclusions
The solution structure of GAIP has been determined using residual dipolar couplings resulting from the alignment of the protein in two distinct liquid crystal media. The orientation of the alignment is different in each medium, therefore, the dipolar couplings vary from the bacteriophage to the bicelle media. Both sets of data agree well with a good precision ensemble of calculated structures, once the axial component and the rhombicity of the respective alignment tensors are properly
Expression, purification and sample preparation
The DNA sequence encoding GAIP primary structure from residue P79 to residue L206 has been inserted in a glutathione S-transferase (GST) fusion vector (pGEX-2T) from Pharmacia Biotech. Escherichia coli BL21(DE3) was used as an expression host. The plasmid once in E. coli was isolated and sequenced and the amino acid sequence of the expressed protein (excluding the GST sequence) is as follows:
Acknowledgements
We are grateful to Dr Hank Fales for analyzing the mass spectra of the protein. We thank Dr Daniel Libutti and Dr Solly Weiler for plasmid sequencing and help with plasmid purification, respectively, and Dr John Louis for providing us with a bacteriophage sample. We are thankful to Dr Frank Delaglio and Dr Dan Garrett for software support and help with NMRPipe/TALOS and PIPP programs, respectively.
E. de A. is recipient of a postdoctoral fellowship from the Human Frontier Science Program.
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