The same mutation in Gs␣ and transducin ␣ reveals behavioral differences between these highly homologous G protein ␣-subunits Adolfo R. Zurita and Lutz Birnbaumer† Laboratory of Neurobiology, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Building 101, Room F180, 111 T. W. Alexander Drive, Research Triangle Park, NC 27709 Contributed by Lutz Birnbaumer, December 27, 2007 (sent for review December 17, 2007) Mutating Arg-238 to Glu (R238E) in the switch 3 region of a unit are largely unknown, and even less well, if at all, understood transducin ␣ (*T␣) in which 27 aa of the GTPase domain have been is how all agonist-occupied (activated) GPCRs promote the replaced with those of the ␣-subunit of the inhibitory G protein 1 same nucleotide exchange reaction at their cognate G protein (Gi1␣), was reported to create an ␣-subunit that is resistant to ␣-subunits. Hypotheses as to how nucleotide exchange comes activation by GTP␥S, is devoid of resident nucleotide, and has about have in common the assumption that similar rate-limiting dominant negative (DN) properties. In an attempt to create a DN steps are facilitated, and that while the receptor–G protein stimultory G protein ␣ (Gs␣) with a single mutation we created interaction is determined by their appropriately differing inter- Gs␣–R265E, equivalent to *T␣–R238E. Gs␣–R265E has facilitated action affinities, the kinetic steps that follow productive associ- activation by GTP␥S, a slightly facilitated activation by GTP but ation of receptor to the trimeric G protein are likely to be very much reduced receptor plus GTP stimulated activation, and an similar, if not the same. apparently unaltered ability to interact with receptor as seen in Transducin, originally called ‘‘light-activated GTPase,’’ was ligand binding studies. Further, the activity profile of Gs␣–R265E is not only the first signal-transducing G protein to be recognized that of an ␣-subunit with unaltered or increased GTPase activity. to have an intrinsic GTPase activity, but also the first for which The only change in Gs␣ that is similar to that in *T␣ is that the ␣␤␥ the nature of its subunit composition was uncovered and the BIOCHEMISTRY apparent affinity for guanine nucleotides is decreased in both first for which it was shown that GTP binding and activation was proteins. The molecular basis of the changed properties are dis- accompanied by an ␣␤␥ to GTP␣ plus ␤␥ dimer subunit cussed based on the known crystal structure of Gs␣ and the dissociation reaction (reviewed in ref. 2). In 1993, T␣ became the changes introduced by the same mutation in a *T␣ (Gt␣*) with only first G␣-subunit for which a crystal-based model became avail- 23 aa from Gi1␣.Gt␣*–R238E, with four fewer mutations in switch able (5, 6). Structures for the ␣-subunit of the inhibitory G 3, was reported to show no evidence of DN properties, is activated protein 1 (Gi1␣) (7), the ␤␥ dimer (8), and trimeric ␣␤␥ forms by GTP␥S, and has reduced GTPase activity. The data highlight a of transducin and Gi1 (9, 10) were reported shortly thereafter. critical role for the switch 3 region in setting overall properties of The ␣-subunits were shown to be two-domain structures: an signal-transducing GTPases. Ϸ180-aa GTPase domain, highly homologous to the smaller regulatory GTPase ras, and, inserted into ras’s switch 1 effector adenylyl cyclase ͉ ␤-adrenergic receptor ͉ GTP shift ͉ GTPase ͉ crystal sequence, a Ϸ120-aa six-helix helical domain (␣A through ␣F) connected at each end to Switch 1 by linkers 1 and 2. Comparison eterotrimeric G proteins are molecular machines that trans- of the structures of ␣-subunits in their inactive, GDP-bound Hduce the signal generated by the binding of agonists to forms (e.g., refs. 9–11) to those of their activated forms, occu- seven-transmembrane receptors into changes in the activity of pied by either GTP␥S (5, 12) or GMP-P(NH)P (13) led to the effectors. Seven transmembrane receptors, also known as G identification of three ‘‘switch’’ regions. G␣ switch 1 and 2 are protein-coupled receptors (GPCRs), and G proteins each con- structurally homologous to the switch regions identified previ- stitute a family of structurally and functionally related molecules. ously in ras (14). Switch 3 comprises the loop connecting the The basic mechanism by which all GPCRs act is by catalyzing the GTPase domain’s fourth ␤-strand (␤4) to its third ␣-helix (␣3). exchange of GTP for GDP on the ␣-subunits of the trimeric G The final ␣5-helix of ␣-subunits is somewhat shorter than ras ␣5 proteins. The binding of the nucleoside triphosphate promotes and includes at its C terminus 10 aa involved in receptor the dissociation of the so-far inactive trimer into an ␣GTP recognition (15). The guanine nucleotide binding site is formed complex plus a ␤␥ dimer, both of which are competent to interact by the GTPase domain, but is somewhat occluded by the closely and modulate the activity states of effectors (for recent reviews juxtaposed helical domain. see refs. 1–3). A strict set of specificities exists that defines which Activation of G␣-subunits (as well as of ras and ras-like of the 16 G protein ␣-subunits and which of the G␤␥ dimers GTPases) involves the binding of both GTP and Mg2ϩ (Fig. 1). interact with which effector function. These specificity rules, Binding of Mg2ϩ involves six coordination bonds of which two which are best understood for ␣-subunits, dictate that the are contributed by GTP (one oxygen each of the ␤ and ␥ activated forms of stimultory G protein ␣ (Gs␣) stimulate phosphates, ␤O and ␥O) and two are provided by water oxygens adenylyl cyclases (ACs), the activated forms of Gi␣ inhibit AC, locked in place by hydrogen bonds, one to the ␦O of an aspartic and the rod and cone transducin ␣-subunits (T␣s) activate visual acid (Asp-223 in Gs␣) and the other to an oxygen of the phosphodiesterase (PDE) in rod and cone photoreceptor cells, ␣-phosphate of the GTP. The last two coordination bonds are respectively. Differences in primary amino acid sequence among provided by oxygens of G␣ amino acids: one of a Ser (Ser-54 of G protein ␣-subunits define their effector specificities. In sup- Gs␣) and the other of a Thr (Thr-204 in Gs␣). port, studies of chimeric ␣-subunits have borne out the assump- tion that effector specificity resides in well defined topologically identified regions of ␣-subunits (cf. ref. 4). Author contributions: A.R.Z. and L.B. designed research; A.R.Z. performed research; A.R.Z. In contrast to the easily understandable differences in effector and L.B. analyzed data; and A.R.Z. and L.B. wrote the paper. specificity, structural features of ␣-subunits that define which of The authors declare no conflict of interest. the many highly homologous GPCRs interacts with which ␣-sub- †To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712261105 PNAS ͉ February 19, 2008 ͉ vol. 105 ͉ no. 7 ͉ 2363–2368 Downloaded by guest on October 2, 2021 Fig. 1. Model of the 3D structure, based on coordinates taken from Protein Data Base ID code 1JCV, of the region of Gs␣ thought to be affected in the Gs␣–R265E mutant. Mg and its six coordination bonds are shown in green; oxygen atoms of two water molecules (w), and relevant oxygens contributing to the coordination shell of Mg are shown in red, as are oxygens of Asp-223 and the ␣ phosphate (␣O), which stabilize by hydrogen bonds the coordinat- ing water molecules. Some secondary structure features of Gs␣ are high- lighted, as are the distances between atoms of Glu-50, Arg-258, and Arg-265 thought to interact by forming ion pairs. Using a chimera between T␣ and Gi1␣ (*Ta), which is better suited for expression in bacteria while preserving all known functions of T␣ (4), as a model to explore the effects of mutations that would be predicted by the crystal structures to affect the interaction between the GTPase and the helical domains, Cerione and collaborators (16, 17) have recently tested how these mutations would correlate with changes in function- ality. One such mutant, *T␣–R238E, which caught our attention Fig. 2. Multiple amino acid sequence alignment comparing bovine trans- as it could not be activated by GTP␥S, was devoid of GDP or ducin ␣ (␣t), human Gi1␣ (␣i), the 394 variant of human Gs␣ (␣s), and *T␣ (chim ␣ ␣ ␣ Ϫ GTP, i.e., it was nucleotide-free, and behaved as a dominant t- i1). Amino acid sequences are compared with that of Gs , where denotes that the amino acid is identical to that in Gs␣. A consensus line is negative (DN) when placed in an assay in which it could compete ␣ shown for which an uppercase letter identifies the identity among all com- with activated WT *T for activation of PDE, the effector of pared sequences, and Ϫ denotes that in at least one of the proteins the transducin (17). R238 of *T␣ lies in the Switch 3 domain of *T␣ sequence differs by one or more amino acids. Secondary structure features (Fig. 2). (␤-strands 1–6, ␣-helices 1–5 and A to G, switch 1, 2, and 3 regions, GTPase to There have been previous attempts to create DN forms of helical domain linkers Lk1 and Lk2) are highlighted, as are sites of ADP- Gs␣. The first was the attempt by Hildebrandt et al. (18) who ribosylation by cholera (CTX) and pertussis (PTX) toxins and interactions with introduced into Gs␣ the Ser-to-Asn mutation that confers DN Mg. The alignment was generated with Accelrys GCG software using PILEUP properties to small ras-like GTPases.