Interaction networks within disease-associated GαS variants characterized by an integrative biophysical approach.

Activation of G proteins through nucleotide exchange initiates intracellular signaling cascades essential for life processes. Under normal conditions, nucleotide exchange is regulated by the formation of G protein-G protein-coupled receptor complexes. Single point mutations in the Gα subunit of G proteins bypass this interaction, leading to loss of function or constitutive gain of function, which is closely linked with the onset of multiple diseases. Despite the recognized significance of Gα mutations in disease pathology, structural information for most variants is lacking, potentially due to inherent protein dynamics that pose challenges for crystallography. To address this, we leveraged an integrative spectroscopic and computational approach to structurally characterize seven of the most frequently observed and clinically relevant mutations in the stimulatory Gα subunit, GαS. A previously proposed allosteric model of Gα activation linked structural changes in the nucleotide-binding pocket with functionally important changes in interactions between switch regions. We investigated this allosteric connection in GαS by integrating data from variable temperature CD spectroscopy, which measured changes in global protein structure and stability, and molecular dynamics simulations, which observed changes in interaction networks between GαS switch regions. Additionally, saturation-transfer difference NMR spectroscopy was applied to observe changes in nucleotide interactions with residues within the nucleotide binding site. These data have enabled testing of predictions regarding how mutations in GαS result in loss or gain of function and evaluation of proposed structural mechanisms. The integration of experimental and computational data allowed us to propose a more nuanced classification of mechanisms underlying GαS gain-of-function and loss-of-function mutations.

Structural and Functional Implication of Natural Variants of Gαs.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are among the most important cellular signaling components, especially G protein-coupled receptors (GPCRs). G proteins comprise three subunits, Gα, Gß, and Gγ. Gα is the key subunit, and its structural state regulates the active status of G proteins. Interaction of guanosine diphosphate (GDP) or guanosine triphosphate (GTP) with Gα switches G protein into basal or active states, respectively. Genetic alteration in Gα could be responsible for the development of various diseases due to its critical role in cell signaling. Specifically, loss-of-function mutations of Gαs are associated with parathyroid hormone-resistant syndrome such as inactivating parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) signaling disorders (iPPSDs), whereas gain-of-function mutations of Gαs are associated with McCune-Albright syndrome and tumor development. In the present study, we analyzed the structural and functional implications of natural variants of the Gαs subtype observed in iPPSDs. Although a few tested natural variants did not alter the structure and function of Gαs, others induced drastic conformational changes in Gαs, resulting in improper folding and aggregation of the proteins. Other natural variants induced only mild conformational changes but altered the GDP/GTP exchange kinetics. Therefore, the results shed light on the relationship between natural variants of Gα and iPPSDs.

Molecular insights into differentiated ligand recognition of the human parathyroid hormone receptor 2.

The parathyroid hormone receptor 2 (PTH2R) is a class B1 G protein-coupled receptor (GPCR) involved in the regulation of calcium transport, nociception mediation, and wound healing. Naturally occurring mutations in PTH2R were reported to cause hereditary diseases, including syndromic short stature. Here, we report the cryogenic electron microscopy structure of PTH2R bound to its endogenous ligand, tuberoinfundibular peptide (TIP39), and a heterotrimeric Gs protein at a global resolution of 2.8 Å. The structure reveals that TIP39 adopts a unique loop conformation at the N terminus and deeply inserts into the orthosteric ligand-binding pocket in the transmembrane domain. Molecular dynamics simulation and site-directed mutagenesis studies uncover the basis of ligand specificity relative to three PTH2R agonists, TIP39, PTH, and PTH-related peptide. We also compare the action of TIP39 with an antagonist lacking six residues from the peptide N terminus, TIP(7-39), which underscores the indispensable role of the N terminus of TIP39 in PTH2R activation. Additionally, we unveil that a disease-associated mutation G258D significantly diminished cAMP accumulation induced by TIP39. Together, these results not only provide structural insights into ligand specificity and receptor activation of class B1 GPCRs but also offer a foundation to systematically rationalize the available pharmacological data to develop therapies for various disorders associated with PTH2R.

Biomarker potential of lncRNA GNAS-AS1 in osteosarcoma prognosis and effect on cellular function.

BACKGROUND: Osteosarcoma (OS) is a type of bone cancer that occurs in children and adolescents at a rate of 5%. The purpose of this study is to explore the lncRNA GNAS-AS1 expression profile, prognosis significance in OS, and biological effect on OS cell function. METHODS: One hundred eight pairs of tissues were collected, and OS cell lines were purchased. lncRNA GNAS-AS1 expression in these tissues and cells were analyzed by qRT-PCR. Clinical data were analyzed using chi-square tests, Kaplan-Meier curves (log-rank test), and Cox regression. CCK-8 and transwell assay were conducted to analyze the effect of lncRNA GNAS-AS1 on cell proliferation, invasion, and migration. The downstream miRNA was presumed. RESULTS: The expression of lncRNA GNAS-AS1 was significantly increased in OS cells and tissues, and related to Enneking staging and distant metastasis. Patients with high lncRNA GNAS-AS1 expression represented shorter overall survival and was an independent prognostic predictor of OS. LncRNA GNAS-AS1 knockdown inhibited cell proliferation, migration, and invasion by regulated miR-490-3p partly at least. CONCLUSIONS: LncRNA GNAS-AS1 can be used as a prognostic indicator and its inhibition suppress the development of OS, suggesting its value as novel therapeutic strategies in OS.

Structure and dynamics of the active Gs-coupled human secretin receptor.

The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Molecular understanding of SecR binding and activation is important for its therapeutic exploitation. We combined cryo-electron microscopy, molecular dynamics, and biochemical cross-linking to determine a 2.3 Å structure, and interrogate dynamics, of secretin bound to the SecR:Gs complex. SecR exhibited a unique organization of its extracellular domain (ECD) relative to its 7-transmembrane (TM) core, forming more extended interactions than other family members. Numerous polar interactions formed between secretin and the receptor extracellular loops (ECLs) and TM helices. Cysteine-cross-linking, cryo-electron microscopy multivariate analysis and molecular dynamics simulations revealed that interactions between peptide and receptor were dynamic, and suggested a model for initial peptide engagement where early interactions between the far N-terminus of the peptide and SecR ECL2 likely occur following initial binding of the peptide C-terminus to the ECD.

Association of Both Inhibitory and Stimulatory Gα Subunits Implies Adenylyl Cyclase 5 Deactivation.

Adenylyl cyclase (AC) generates cyclic AMP required for a variety of cellular functions, and its regulation plays a major role in cellular signal transduction in eukaryotes and prokaryotes. All membrane-bound AC isoforms in eukaryotes can be activated by stimulatory G-proteins, but only AC1, AC5, and AC6 can be both stimulated and inhibited by active Gα subunits, Gαs and Gαi, respectively. In principle, these Gαi-sensitive AC isoforms could form both binary and ternary complexes with Gα subunits due to the noncompetitive association of inhibitory and stimulatory Gα. However, the formation and possible catalytic activity of a putative ternary complex have not yet been experimentally confirmed due to its proposed short-lived nature. Here, the catalytic activity of such a ternary complex consisting of apo AC5, stimulatory Gαolf, and inhibitory Gαi1 is investigated via classical molecular dynamics simulations. Trajectories of inhibited and stimulated binary complexes, AC5:Gαi1 and AC5:Gαolf, respectively, as well as Gα-free AC5 were also obtained to compare the sampled AC5 conformation in the ternary complex to those sampled under different Gα conditions. This comparison suggests that association of both Gα subunits results in an AC5 conformation similar to that sampled by the AC5:Gαi1 complex, indicating that the ternary complex mainly samples an inactive conformation.

The structure of a membrane adenylyl cyclase bound to an activated stimulatory G protein.

Membrane-integral adenylyl cyclases (ACs) are key enzymes in mammalian heterotrimeric GTP-binding protein (G protein)-dependent signal transduction, which is important in many cellular processes. Signals received by the G protein-coupled receptors are conveyed to ACs through G proteins to modulate the levels of cellular cyclic adenosine monophosphate (cAMP). Here, we describe the cryo-electron microscopy structure of the bovine membrane AC9 bound to an activated G protein αs subunit at 3.4-angstrom resolution. The structure reveals the organization of the membrane domain and helical domain that spans between the membrane and catalytic domains of AC9. The carboxyl-terminal extension of the catalytic domain occludes both the catalytic and the allosteric sites of AC9, inducing a conformation distinct from the substrate- and activator-bound state, suggesting a regulatory role in cAMP production.

Structure of the adenosine-bound human adenosine A1 receptor-Gi complex.

The class A adenosine A1 receptor (A1R) is a G-protein-coupled receptor that preferentially couples to inhibitory Gi/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A1R in complex with adenosine and heterotrimeric Gi2 protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive A1R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A1R primarily via amino acids in the C terminus of the Gαi α5-helix, concomitant with a 10.5 Å outward movement of the A1R transmembrane domain 6. Comparison with the agonist-bound ß2 adrenergic receptor-Gs-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A1R structure provides molecular insights into receptor and G-protein selectivity.

Cryo-EM structure of the serotonin 5-HT1B receptor coupled to heterotrimeric Go.

G-protein-coupled receptors (GPCRs) form the largest family of receptors encoded by the human genome (around 800 genes). They transduce signals by coupling to a small number of heterotrimeric G proteins (16 genes encoding different α-subunits). Each human cell contains several GPCRs and G proteins. The structural determinants of coupling of Gs to four different GPCRs have been elucidated1-4, but the molecular details of how the other G-protein classes couple to GPCRs are unknown. Here we present the cryo-electron microscopy structure of the serotonin 5-HT1B receptor (5-HT1BR) bound to the agonist donitriptan and coupled to an engineered Go heterotrimer. In this complex, 5-HT1BR is in an active state; the intracellular domain of the receptor is in a similar conformation to that observed for the ß2-adrenoceptor (ß2AR) 3 or the adenosine A2A receptor (A2AR) 1 in complex with Gs. In contrast to the complexes with Gs, the gap between the receptor and the Gß-subunit in the Go-5-HT1BR complex precludes molecular contacts, and the interface between the Gα-subunit of Go and the receptor is considerably smaller. These differences are likely to be caused by the differences in the interactions with the C terminus of the Go α-subunit. The molecular variations between the interfaces of Go and Gs in complex with GPCRs may contribute substantially to both the specificity of coupling and the kinetics of signalling.

Disease-Causing Mutations in the G Protein Gαs Subvert the Roles of GDP and GTP.

The single most frequent cancer-causing mutation across all heterotrimeric G proteins is R201C in Gαs. The current model explaining the gain-of-function activity of the R201 mutations is through the loss of GTPase activity and resulting inability to switch off to the GDP state. Here, we find that the R201C mutation can bypass the need for GTP binding by directly activating GDP-bound Gαs through stabilization of an intramolecular hydrogen bond network. Having found that a gain-of-function mutation can convert GDP into an activator, we postulated that a reciprocal mutation might disrupt the normal role of GTP. Indeed, we found R228C, a loss-of-function mutation in Gαs that causes pseudohypoparathyroidism type 1a (PHP-Ia), compromised the adenylyl cyclase-activating activity of Gαs bound to a non-hydrolyzable GTP analog. These findings show that disease-causing mutations in Gαs can subvert the canonical roles of GDP and GTP, providing new insights into the regulation mechanism of G proteins.

Cross-communication between Gi and Gs in a G-protein-coupled receptor heterotetramer guided by a receptor C-terminal domain.

BACKGROUND: G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (A1R or A2AR) can form A1R-A2AR heteromers (A1-A2AHet), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (ß-arrestin mediated) signaling. Adenosine has different affinities for A1R and A2AR, allowing the heteromeric receptor to detect its concentration by integrating the downstream Gi- and Gs-dependent signals. cAMP accumulation and ß-arrestin recruitment assays have shown that, within the complex, activation of A2AR impedes signaling via A1R. RESULTS: We examined the mechanism by which A1-A2AHet integrates Gi- and Gs-dependent signals. A1R blockade by A2AR in the A1-A2AHet is not observed in the absence of A2AR activation by agonists, in the absence of the C-terminal domain of A2AR, or in the presence of synthetic peptides that disrupt the heteromer interface of A1-A2AHet, indicating that signaling mediated by A1R and A2AR is controlled by both Gi and Gs proteins. CONCLUSIONS: We identified a new mechanism of signal transduction that implies a cross-communication between Gi and Gs proteins guided by the C-terminal tail of the A2AR. This mechanism provides the molecular basis for the operation of the A1-A2AHet as an adenosine concentration-sensing device that modulates the signals originating at both A1R and A2AR.

Methylglyoxal Impairs ß2-Adrenoceptor-Mediated Vasodilatory Mechanisms in Rat Retinal Arterioles.

Methylglyoxal, a highly reactive dicarbonyl compound, is formed as a by-product of glycolysis and plays an important role in the pathogenesis of diabetic complications, including diabetic retinopathy. However, it remains to be determined how methylglyoxal affects the regulatory mechanisms of retinal blood flow. In this study, we examined the effects of methylglyoxal on ß2-adrenoceptor-mediated vasodilatory mechanisms in rat retinal arterioles. The retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles in the fundus images. Intravitreal injection of methylglyoxal significantly diminished the vasodilation of retinal arterioles induced by the ß2-adrenoceptor agonist salbutamol. The vasodilator effect of BMS-191011, a large-conductance Ca2+-activated K+ (BKCa) channel opener, on retinal arterioles was also attenuated by methylglyoxal. In contrast, methylglyoxal had no significant effect on retinal vasodilator response to forskolin. Methylglyoxal attenuated retinal vasodilator response to salbutamol under blockade of BKCa channels with iberiotoxin, an inhibitor of the channels. These results suggest that methylglyoxal attenuates ß2-adrenoceptor-mediated retinal vasodilation by impairing the coupling of the ß2-adrenoceptor to the guanine nucleotide-binding protein (Gs protein) and the function of the BKCa channel. Increased methylglyoxal in the eyes may contribute to the impairment of regulatory mechanisms of retinal blood flow in patients with diabetic retinopathy.

Study of adenylyl cyclase-GαS interactions and identification of novel AC ligands.

Adenylyl cyclases (ACs) are membrane bound enzymes that catalyze the production of cAMP from ATP in response to the activation by G-protein Gαs. Different isoforms of ACs are ubiquitously expressed in different tissues involved in regulatory mechanisms in response to specific stimulants. There are 9 AC isoforms present in humans, with AC5 and AC6 proposed to play a vital role in cardiac functions. The activity of AC6 is sensitive to nitric oxide, such that nitrosylation of the protein might regulate its function. However, the information on structural determinants of nitrosylation in ACs and how they interact with Gαs is limited. Here we used homology modeling to build a molecular model of human AC6 bound to Gαs. Based on this 3D model, we predict the nitrosylation amenable cysteines, and identify potential novel ligands of AC6 using virtual ligand screening. Our model suggests Cys1004 in AC6 (subunit C2) and Cys174 in Gαs present at the AC-Gαs interface as the possible residues that might undergo reversible nitrosylation. Docking analysis predicted novel ligands of AC6 that include forskolin-based compounds and its derivatives. Further work involving site-directed mutagenesis of the predicted residues will allow manipulation of AC activity using novel ligands, and crucial insights on the role of nitrosylation of these proteins in pathophysiological conditions.

Gαs protein binds ubiquitin to regulate epidermal growth factor receptor endosomal sorting.

The Gαs subunit is classically involved in the signal transduction of G protein-coupled receptors (GPCRs) at the plasma membrane. Recent evidence has revealed noncanonical roles for Gαs in endosomal sorting of receptors to lysosomes. However, the mechanism of action of Gαs in this sorting step is still poorly characterized. Here, we report that Gαs interacts with ubiquitin to regulate the endosomal sorting of receptors for lysosomal degradation. We reveal that the N-terminal extremity of Gαs contains a ubiquitin-interacting motif (UIM), a sorting element usually found in the endosomal sorting complex required for transport (ESCRT) machinery responsible for sorting ubiquitinated receptors into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). Mutation of the UIM in Gαs confirmed the importance of ubiquitin interaction for the sorting of epidermal growth factor receptor (EGFR) into ILVs for lysosomal degradation. These findings demonstrate a role for Gαs as an integral component of the ubiquitin-dependent endosomal sorting machinery and highlight the dual role of Gαs in receptor trafficking and signaling for the fine-tuning of the cellular response.

Strategy for the Thermostabilization of an Agonist-Bound GPCR Coupled to a G Protein.

Structure determination of G protein-coupled receptors (GPCRs) in the inactive state bound to high-affinity antagonists has been very successful through the implementation of a number of protein engineering and crystallization strategies. However, the structure determination of GPCRs in their fully active state coupled to a G protein is still very challenging. Recently, mini-G proteins were developed, which recapitulate the coupling of a full heterotrimeric G protein to a GPCR despite being less than one-third of the size. This allowed the structure determination of the agonist-bound adenosine A2A receptor (A2AR) coupled to mini-Gs. Although this is extremely encouraging, A2AR is very stable compared with many other GPCRs, particularly when an agonist is bound. In contrast, the agonist-bound conformation of the human corticotropin-releasing factor receptor is considerably less stable, impeding the formation of good quality crystals for structure determination. We have therefore developed a novel strategy for the thermostabilization of a GPCR-mini-G protein complex. In this chapter, we will describe the theoretical and practical principles of the thermostability assay for stabilizing this complex, discuss its strengths and weaknesses, and show some typical results from the thermostabilization process.

Internalized Receptor for Glucose-dependent Insulinotropic Peptide stimulates adenylyl cyclase on early endosomes.

Until very recently, G-protein dependent signal of GPCRs was thought to originate exclusively from the plasma membrane and internalized GPCRs were considered silent. Here, we demonstrated that, once internalized and located in the membrane of early endosomes, glucose-dependent Insulinotropic receptor (GIPR) continues to trigger production of cAMP and PKA activation. Direct evidence is based on identification of the active form of Gαs in early endosomes containing GIPR using a genetically encoded GFP tagged nanobody, and on detection of a distinct FRET signal accounting for cAMP production at the surface of endosomes containing GIP, compared to endosomes without GIP. Furthermore, decrease of the sustained phase of cAMP production and PKA activation kinetics as well as reversibility of cAMP production and PKA activity following GIP washout in cells treated with a pharmacological inhibitor of GIPR internalization, and continuous increase of cAMP level over time in the presence of dominant-negative Rab7, which causes accumulation of early endosomes in cells, were noticed. Hence the GIPR joins the few GPCRs which signal through G-proteins both at plasma membrane and on endosomes.

Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling.

Although the importance of the C terminus of the α subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the Gα subunit (Gα peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between Gα peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate Gα peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate Gα peptide stabilizes the agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gαs/Gαq-Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the ß2-adrenergic receptor (ß2-AR)-Gαs and V1A receptor (V1AR)-Gαq The Gαs and Gαq peptides adopt different orientations in ß2-AR and V1AR, respectively. The ß2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gαq peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in Gαq prevents this peptide from interacting strongly with ß2-AR. Mutagenesis to the Gαs counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions.

International Union of Basic and Clinical Pharmacology. XCIII. The parathyroid hormone receptors--family B G protein-coupled receptors.

The type-1 parathyroid hormone receptor (PTHR1) is a family B G protein-coupled receptor (GPCR) that mediates the actions of two polypeptide ligands; parathyroid hormone (PTH), an endocrine hormone that regulates the levels of calcium and inorganic phosphate in the blood by acting on bone and kidney, and PTH-related protein (PTHrP), a paracrine-factor that regulates cell differentiation and proliferation programs in developing bone and other tissues. The type-2 parathyroid hormone receptor (PTHR2) binds a peptide ligand, called tuberoinfundibular peptide-39 (TIP39), and while the biologic role of the PTHR2/TIP39 system is not as defined as that of the PTHR1, it likely plays a role in the central nervous system as well as in spermatogenesis. Mechanisms of action at these receptors have been explored through a variety of pharmacological and biochemical approaches, and the data obtained support a basic "two-site" mode of ligand binding now thought to be used by each of the family B peptide hormone GPCRs. Recent crystallographic studies on the family B GPCRs are providing new insights that help to further refine the specifics of the overall receptor architecture and modes of ligand docking. One intriguing pharmacological finding for the PTHR1 is that it can form surprisingly stable complexes with certain PTH/PTHrP ligand analogs and thereby mediate markedly prolonged cell signaling responses that persist even when the bulk of the complexes are found in internalized vesicles. The PTHR1 thus appears to be able to activate the Gα(s)/cAMP pathway not only from the plasma membrane but also from the endosomal domain. The cumulative findings could have an impact on efforts to develop new drug therapies for the PTH receptors.

Familial pituitary tumors.

Pituitary adenomas are benign intracranial neoplasms that present a major clinical concern due to hormone overproduction and/or tumor mass effects. The majority of pituitary adenomas occur sporadically; however, familial cases are increasingly being recognized, such as multiple endocrine neoplasia type 1 (MEN1), Carney complex (CNC), and familial isolated pituitary adenoma (FIPA). Familial pituitary tumors appear to differ from their sporadic counterparts both in their genetic basis and in clinical characteristics. Evidence suggests that, especially in MEN1 and FIPA, tumors are more aggressive and affect patients at a younger age, therefore justifying the importance of early diagnosis, while in Carney complex pituitary hyperplasia is common. The genetic alterations responsible for the formation of familial pituitary syndromes include the MEN1 gene, responsible for about 80% of MEN1 cases, the regulatory subunit of the protein kinase A, PRKAR1A, responsible for about 70% of Carney complex cases, and AIP, the gene coding the aryl hydrocarbon receptor interacting protein, responsible for about 20% of FIPA cases. Rarely other genes have also been found responsible for familial pituitary adenoma cases. McCune-Albright syndrome (MAS) also has a genetic origin due to mosaic mutations in the G protein-coupled α subunit coded by the GNAS1 gene. In this chapter, we summarize the genetic and clinical characteristics of these familial pituitary syndromes and MAS.

Different biochemical properties explain why two equivalent Gα subunit mutants cause unrelated diseases.

There is an increasing number of disease-associated Gα mutations identified from genome-wide sequencing campaigns or targeted efforts. Albright's Hereditary Osteodystrophy (AHO) was the first inherited disease associated with loss-of-function mutations in a G protein (Gαs) and other studies revealed gain-of-function Gα mutations in cancer. Here we attempted to solve the apparent quandary posed by the fact that the same mutation in two different G proteins appeared associated with both AHO and cancer. We first confirmed the presence of an inherited Gαs-R265H mutation from a previously described clinical case report of AHO. This mutation is structurally analogous to Gαo-R243H, an oncogenic mutant with increased activity in vitro and in cells due to rapid nucleotide exchange. We found that, contrary to Gαo-R243H, Gαs-R265H activity is compromised due to greatly impaired nucleotide binding in vitro and in cells. We obtained equivalent results when comparing another AHO mutation in Gαs (D173N) with a counterpart cancer mutation in Gαo (D151N). Gαo-R243H binds nucleotides efficiently under steady-state conditions but releases GDP much faster than the WT protein, suggesting diminished affinity for the nucleotide. These results indicate that the same disease-linked mutation in two different G proteins affects a common biochemical feature (nucleotide affinity) but to a different grade depending on the G protein (mild decrease for Gαo and severe for Gαs). We conclude that Gαs-R265H has dramatically impaired nucleotide affinity leading to the loss-of-function in AHO whereas Gαo-R243H has a mild decrease in nucleotide affinity that causes rapid nucleotide turnover and subsequent hyperactivity in cancer.