Molecular mechanism of regulation of RhoA GTPase by phosphorylation of RhoGDI.

Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) play a crucial role in the regulation of Rho family GTPases. They act as negative regulators that prevent the activation of Rho GTPases by forming complexes with the inactive GDP-bound state of GTPase. Release of Rho GTPase from the RhoGDI-bound complex is necessary for Rho GTPase activation. Biochemical studies provide evidence of a "phosphorylation code," where phosphorylation of some specific residues of RhoGDI selectively releases its GTPase partner (RhoA, Rac1, Cdc42, etc.). This work attempts to understand the molecular mechanism behind this specific phosphorylation-induced reduction in binding affinity. Using several microseconds long atomistic molecular dynamics simulations of the wild-type and phosphorylated states of the RhoA-RhoGDI complex, we propose a molecular-interaction-based mechanistic model for the dissociation of the complex. Phosphorylation induces major structural changes, particularly in the positively charged polybasic region (PBR) of RhoA and the negatively charged N-terminal region of RhoGDI that contribute most to the binding affinity. Molecular mechanics Poisson-Boltzmann surface area binding energy calculations show a significant weakening of interaction on phosphorylation at the RhoA-specific site of RhoGDI. In contrast, phosphorylation at a Rac1-specific site does not affect the overall binding affinity significantly, which confirms the presence of a phosphorylation code. RhoA-specific phosphorylation leads to a reduction in the number of contacts between the PBR of RhoA and the N-terminal region of RhoGDI, which manifests a reduction of the binding affinity. Using hydrogen bond occupancy analysis and energetic perturbation network, we propose a mechanistic model for the allosteric response, i.e., long-range signal propagation from the site of phosphorylation to the PBR and buried geranylgeranyl group in the form of rearrangement and rewiring of hydrogen bonds and salt bridges. Our results highlight the crucial role of specific electrostatic interactions in manifestation of the phosphorylation code.

RhoA regulation in space and time.

RhoGTPases are well known for being controllers of cell cytoskeleton and share common features in the way they act and are controlled. These include their switch from GDP to GTP states, their regulations by different guanine exchange factors (GEFs), GTPase-activating proteins and guanosine dissociation inhibitors (GDIs), and their similar structure of active sites/membrane anchors. These very similar features often lead to the common consideration that the differences in their biological effects mainly arise from the different types of regulators and specific effectors associated with each GTPase. Focusing on data obtained through biosensors, live cell microscopy and recent optogenetic approaches, we highlight in this review that the regulation of RhoA appears to depart from Cdc42 and Rac1 modes of regulation through its enhanced lability at the plasma membrane. RhoA presents a high dynamic turnover at the membrane that is regulated not only by GDIs but also by GEFs, effectors and a possible soluble conformational state. This peculiarity of RhoA regulation may be important for the specificities of its functions, such as the existence of activity waves or its putative dual role in the initiation of protrusions and contractions.

GPSM1 impairs metabolic homeostasis by controlling a pro-inflammatory pathway in macrophages.

G-protein-signaling modulator 1 (GPSM1) exhibits strong genetic association with Type 2 diabetes (T2D) and Body Mass Index in population studies. However, how GPSM1 carries out such control and in which types of cells are poorly understood. Here, we demonstrate that myeloid GPSM1 promotes metabolic inflammation to accelerate T2D and obesity development. Mice with myeloid-specific GPSM1 ablation are protected against high fat diet-induced insulin resistance, glucose dysregulation, and liver steatosis via repression of adipose tissue pro-inflammatory states. Mechanistically, GPSM1 deficiency mainly promotes TNFAIP3 transcription via the Gαi3/cAMP/PKA/CREB axis, thus inhibiting TLR4-induced NF-κB signaling in macrophages. In addition, we identify a small-molecule compound, AN-465/42243987, which suppresses the pro-inflammatory phenotype by inhibiting GPSM1 function, which could make it a candidate for metabolic therapy. Furthermore, GPSM1 expression is upregulated in visceral fat of individuals with obesity and is correlated with clinical metabolic traits. Overall, our findings identify macrophage GPSM1 as a link between metabolic inflammation and systemic homeostasis.

The atypical Rab GTPase associated with Parkinson's disease, Rab29, is localized to membranes.

Several genetic studies have shown that the small GTPase Rab29 is involved in the pathogenesis of Parkinson's Disease (PD). It has also been shown that overexpression of Rab29 increases the activity of leucine-rich repeat kinase 2, a protein kinase often mutated in familial PD, although the mechanism underlying this activation remains unclear. Here, we employed biochemical analyses to characterize the localization of Rab29 and found that, unlike general Rab proteins, Rab29 is predominantly fractionated into the membrane fraction by ultracentrifugation. We also found that Rab29 is resistant to extraction from membranes by GDP-dissociation inhibitors (GDIs) in vitro. Furthermore, Rab29 failed to interact with GDIs, and its membrane localization was not affected by the knockout of GDIs in cells. We show that the knockout of Rab geranylgeranyltransferase decreased the hydrophobicity of Rab29, suggesting that Rab29 is geranylgeranylated at its carboxyl terminus as is with typical Rab proteins. Notably, we demonstrated that membrane-bound Rab29 retains some hydrophilicity, indicating that mechanisms other than geranylgeranylation might also be involved in the membrane localization of Rab29. Taken together, these findings uncover the atypical nature of Rab29 among Rab proteins, which will provide important clues for understanding how Rab29 is involved in the molecular pathomechanism of PD.

GDI2 is a novel diagnostic and prognostic biomarker in hepatocellular carcinoma.

BACKGROUND: GDP Dissociation inhibitor 2 (GDI2) gene has been correlated with some important biological processes in a variety of cancers, whereas the role of GDI2 in hepatocellular carcinoma (HCC) is ill-defined. We aimed to demonstrate the relationship between GDI2 and HCC based on The Cancer Genome Atlas (TCGA) data mining. METHODS: The expression of GDI2 was compared between cancer and normal tissues of 371 HCC patients collected from TCGA-LIHC, and verified in HCC cell lines. Gene set enrichment analysis (GSEA) was applied to annotate biological function of GDI2. Furthermore, Wilcoxon rank sum test, Logistics regression, as well as Cox regression and Kaplan-Meier survival analysis, were employed to evaluate the association of GDI2 expression with clinicopathological characteristics, and survival status of HCC patients, respectively. RESULTS: It showed that the expression of GDI2 was much higher in tumor tissues than in normal tissues (P < 0.001) of HCC patients. And the elevated expression of GDI2 was correlated with more aggressive HCC tumor status, including severe primary tumor extent, advanced pathological stage, serious histologic grade, and mutated TP53 status (P < 0.05). Moreover, high GDI2 expression was strongly associated with a poor survival rate (P < 0.001). Both enrichment and immune infiltration analyses implied that GDI2-associated signaling mainly involve lipid metabolism and extracellular matrix (ECM) constructing pathways related to tumor microenvironment (TME) (P < 0.05). CONCLUSIONS: The elevated expression of GDI2 predicts poor prognosis in HCC patients, indicating that GDI2 could be applied as a predictive biomarker for diagnosis and prognosis of HCC.

Overexpression of GDP dissociation inhibitor 1 gene associates with the invasiveness and poor outcomes of colorectal cancer.

GDP dissociation inhibitor (GDI) regulates the GDP/GTP exchange reaction of most Rab proteins by inhibiting GDP dissociation. This study evaluated the potential prognostic and predictive value of GDI1 in colorectal cancer (CRC). To address the prognostic power of GDI1, we performed individual and pooled survival analyses on six independent CRC microarray gene expression datasets. GDI1-enriched signatures were also analyzed. Kaplan-Meier and Cox proportional analyses were employed for survival analysis. An immunohistochemistry (IHC) analysis was performed to validate the clinical relevance and prognostic significance of the GDI1 protein level in CRC tissue samples. The results revealed that GDI1 mRNA level was significantly linked with the aggressiveness of CRC, which is compatible with gene set enrichment analysis. A meta-analysis and pooled analysis demonstrated that a higher mRNA GDI1 expression was dramatically correlated with a worse survival in a dose-dependent manner in CRC patients. Further IHC analysis validated that the protein expression of GDI1 in both cytoplasm and membrane also significantly impacted the outcome of CRC patients. In CRC patients with stage III, chemotherapy significantly reduced the relative risk of death in low-GDI1 subgroup (hazard ratio (HR) = 0.22; 95% confidence interval (95% CI) 0.09-0.56, p = 0.0003), but not in high-GDI1 subgroup (HR = 0.63; 95% CI 0.35-1.14, p = 0.1137). Therefore, both high mRNA and protein levels of GDI1 were significantly related to poor outcomes in CRC patients. GD11 may serve as a prognostic biomarker for CRC.

ISGylation drives basal breast tumour progression by promoting EGFR recycling and Akt signalling.

ISG15 is an ubiquitin-like modifier that is associated with reduced survival rates in breast cancer patients. The mechanism by which ISG15 achieves this however remains elusive. We demonstrate that modification of Rab GDP-Dissociation Inhibitor Beta (GDI2) by ISG15 (ISGylation) alters endocytic recycling of the EGF receptor (EGFR) in non-interferon stimulated cells using CRISPR-knock out models for ISGylation. By regulating EGFR trafficking, ISGylation enhances EGFR recycling and sustains Akt-signalling. We further show that Akt signalling positively correlates with levels of ISG15 and its E2-ligase in basal breast cancer cohorts, confirming the link between ISGylation and Akt signalling in human tumours. Persistent and enhanced Akt activation explains the more aggressive tumour behaviour observed in human breast cancers. We show that ISGylation can act as a driver of tumour progression rather than merely being a bystander.

Imaging of Spatial Cycling of Rab GTPase in the Cell.

Rab GTPases (>60 members in human) function as master regulators of intracellular membrane trafficking. To fulfill their functions, Rab proteins need to localize on specific membranes in cells. It remains elusive how the distinct spatial distribution of Rab GTPases in the cell is regulated. To make a global assessment on the subcellular localization of Rab1, we determined kinetic parameters of the spatial cycling of Rab1 in live cells using photoactivatable fluorescent proteins and live cell imaging. We found that the switching between GTP- and GDP-binding states, which is governed by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), GDP dissociation inhibitor (GDI) and GDI displacement factor (GDF), is a major determinant for Rab1's ability to effectively cycle between cellular compartments and eventually for its subcellular distribution. Herein, we describe the method for monitoring Rab1 dynamics in live cells. This approach can be used to study spatial cycling of other Rab GTPases.

Knockdown of GPSM1 Inhibits the Proliferation and Promotes the Apoptosis of B-Cell Acute Lymphoblastic Leukemia Cells by Suppressing the ADCY6-RAPGEF3-JNK Signaling Pathway.

B-cell acute lymphoblastic leukemia (B-ALL) is the common type of blood cancer. Although the remission rate has increased, the current treatment options for B-ALL are usually related to adverse reactions and recurrence, so it is necessary to find other treatment options. G protein signaling modulator 1 (GPSM1) is one of several factors that affect the basic activity of the G protein signaling system, but its role in B-ALL has not yet been clarified. In this study, we analyzed the expression of GPSM1 in the Oncomine database and found that the GPSM1 levels were higher in B-ALL cells than in peripheral blood mononuclear cells (PBMCs). Analyses of the Gene Expression Profiling Interactive Analysis (GEPIA) demonstrated that patients with high GPSM1 levels had shorter survival times than those with low levels. Additionally, gene set enrichment analysis (GSEA) suggested that GPSM1 was positively correlated with proliferation, G protein-coupled receptor (GPCR) ligand binding, Gαs signaling and calcium signaling pathways. In further experiments, GPSM1 was found to be highly expressed in Acute lymphoblastic leukemia (ALL) cell lines, and downregulation of GPSM1 inhibited proliferation and promoted cell cycle arrest and apoptosis in BALL-1 and Reh cells. Moreover, knockdown of GPSM1 suppressed ADCY6 and RAPGEF3 expression in BALL-1 and Reh cells. Furthermore, we reported that GPSM1 regulated JNK expression via ADCY6-RAPGEF3. The present study demonstrates that GPSM1 promotes tumor growth in BALL-1 and Reh cells by modulating ADCY6-RAPGEF3-JNK signaling.

GDI2 is a target of paclitaxel that affects tumorigenesis of prostate cancer via the p75NTR signaling pathway.

BACKGROUND: Prostate cancer (PCa) refers to malignant tumors derived from prostate epithelial cells, whose morbidity and mortality rates have been increasing every year. Although new drugs for treating prostate cancer continue to emerge, the unclear mechanism underlying drug targets limits this therapy, thereby constraining identification of effective therapeutic targets. Although GDP dissociation inhibitor 2(GDI2) is highly expressed and closely associated with occurrence and development of many tumors, its role in prostate cancer remains unclear. In this study, we investigated the role of GDI2 and elucidated its underlying mechanism of action in prostate cancer. Moreover, we screened chemotherapeutic drugs that affect GDI2 expression with a view of identifying novel targets for diagnosis and treatment of prostate cancer. METHODS: We performed sequence analyses and functional assays to precisely elucidate the GDI2 role in prostate cancer. Moreover, we induced tumorigenesis in nude mice to verify the role of GDI2 in vivo. Finally, we used the CCK8 assay to ascertain the most suitable IC50 across the three drugs and performed quantitative real time polymerase chain reaction (qRT-PCR) and Western Blot to analyze the effects of drugs on expression of GDI2, p75NTR, and p-NFκB. RESULTS: GDI2 was up-regulated in prostate cancer cells and tissues. Knocking down GDI2 suppressed cell proliferation but promoted cell apoptosis. Interestingly, knocking down GDI2 activated the p75NTR signaling pathway, indicating, for the first time, that p75NTR is negatively correlated with GDI2 expression. CONCLUSION: Taken together, these results indicate that GDI2 is a therapeutic target of paclitaxel. Knocking down of GDI2 inhibits cell proliferation and promotes cell apoptosis via the p75NTR signaling pathway in prostate cancer. Notably, paclitaxel inhibits GDI2 expression, implying that GDI2 may be a promising therapeutic target in prostate cancer.

AGS3 and Gαi3 Are Concomitantly Upregulated as Part of the Spindle Orientation Complex during Differentiation of Human Neural Progenitor Cells.

Adult neurogenesis is modulated by many Gi-coupled receptors but the precise mechanism remains elusive. A key step for maintaining the population of neural stem cells in the adult is asymmetric cell division (ACD), a process which entails the formation of two evolutionarily conserved protein complexes that establish the cell polarity and spindle orientation. Since ACD is extremely difficult to monitor in stratified tissues such as the vertebrate brain, we employed human neural progenitor cell lines to examine the regulation of the polarity and spindle orientation complexes during neuronal differentiation. Several components of the spindle orientation complex, but not those of the polarity complex, were upregulated upon differentiation of ENStem-A and ReNcell VM neural progenitor cells. Increased expression of nuclear mitotic apparatus (NuMA), Gαi subunit, and activators of G protein signaling (AGS3 and LGN) coincided with the appearance of a neuronal marker (ß-III tubulin) and the concomitant loss of neural progenitor cell markers (nestin and Sox-2). Co-immunoprecipitation assays demonstrated that both Gαi3 and NuMA were associated with AGS3 in differentiated ENStem-A cells. Interestingly, AGS3 appeared to preferentially interact with Gαi3 in ENStem-A cells, and this specificity for Gαi3 was recapitulated in co-immunoprecipitation experiments using HEK293 cells transiently overexpressing GST-tagged AGS3 and different Gαi subunits. Moreover, the binding of Gαi3 to AGS3 was suppressed by GTPγS and pertussis toxin. Disruption of AGS3/Gαi3 interaction by pertussis toxin indicates that AGS3 may recognize the same site on the Gα subunit as G protein-coupled receptors. Regulatory mechanisms controlling the formation of spindle orientation complex may provide novel means to manipulate ACD which in turn may have an impact on neurogenesis.

Depletion of GPSM1 enhances ovarian granulosa cell apoptosis via cAMP-PKA-CREB pathway in vitro.

BACKGROUND: Genetic causes of premature ovarian insufficiency (POI) account for approximately 20 ~ 25% of patients. So far, only a few genes have been identified. RESULTS: Here, we first identified the c.1840C > A on G-protein signaling modulator 1 (GPSM1) as a susceptibility locus for POI in 10 sporadic POI patients by whole-exome sequencing. The frequency of GPSM1 c.1840C > A was then verified as 3/20 in a POI sample of 20 patients (including the above 10 patients) by Sanger sequencing. RT-PCR and western blot analysis showed the expression of GPSM1 in rat ovaries was increased in the large antral follicle stage compared to the primordial follicle stage (P < 0.01). The cell proliferation assay (CCK8) and flow cytometry suggested that the small-interfering RNA-induced silencing of Gpsm1 significantly increased apoptosis and decreased proliferation of rat ovarian granulosa cells (GCs) (P < 0.01). Furthermore, suppression of Gpsm1 in GCs reduced levels of cAMP, PKAc, p-CREB as well as the ratio of Bcl-2/Bax, and increased the expression of Caspase-3 and Cleaved Caspase-3 (P < 0.01). CONCLUSIONS: In summary, this study identified a susceptibility variant GPSM1 c.1840C > A of POI for the first time. Gpsm1 was related to rat follicle development, and silencing of Gpsm1 increased apoptosis and decreased proliferation in rat GCs, possibly through inhibition of the cAMP-PKA-CREB pathway.

Adipose tissue depot-specific intracellular and extracellular cues contributing to insulin resistance in obese individuals.

Adipose tissue dysregulation in obesity strongly influences systemic metabolic homeostasis and is often linked to insulin resistance (IR). However, the molecular mechanisms underlying adipose tissue dysfunction in obesity are not fully understood. Herein, a proteomic analysis of subcutaneous (SC) and omental (OM) fat from lean subjects and obese individuals with different degrees of insulin sensitivity was performed to identify adipose tissue biomarkers related to obesity-associated metabolic disease. Our results suggest that dysregulation of both adipose tissue extracellular matrix (ECM) organization and intracellular trafficking processes may be associated with IR in obesity. Thus, abnormal accumulation of the small leucine-rich proteoglycan, lumican, as observed in SC fat of IR obese individuals, modifies collagen I organization, impairs adipogenesis and activates stress processes [endoplasmic reticulum and oxidative stress] in adipocytes. In OM fat, IR is associated with increased levels of the negative regulator of the Rab family of small GTPases, GDI2, which alters lipid storage in adipocytes by inhibiting insulin-stimulated binding of the Rab protein, Rab18, to lipid droplets. Together, these results indicate that lumican and GDI2 might play depot-dependent, pathogenic roles in obesity-associated IR. Our findings provide novel insights into the differential maladaptive responses of SC and OM adipose tissue linking obesity to IR.

Genome-wide meta-analysis associates GPSM1 with type 2 diabetes, a plausible gene involved in skeletal muscle function.

Genome-wide association studies (GWASs) have identified many genetic variations associated with type 2 diabetes mellitus (T2DM) in Asians, but understanding the functional genetic variants that influence traits is often a complex process. In this study, fine mapping and other analytical strategies were performed to investigate the effects of G protein signaling modulator 1 (GPSM1) on insulin resistance in skeletal muscle. A total of 128 single-nucleotide polymorphisms (SNPs) within GPSM1 were analysed in 21,897 T2DM cases and 32,710 healthy controls from seven GWASs. The SNP rs28539249 in intron 9 of GPSM1 showed a nominally significant association with T2DM in Asians (OR = 1.07, 95% CI = 1.04-1.10, P < 10-4). The GPSM1 mRNA was increased in skeletal muscle and correlated with T2DM traits across obese mice model. An eQTL for the cis-acting regulation of GPSM1 expression in human skeletal muscle was identified for rs28539249, and the increased GPSM1 expression related with T2DM traits within GEO datasets. Another independent Asian cohort showed that rs28539249 is associated with the skeletal muscle expression of CACFD1, GTF3C5, SARDH, and FAM163B genes, which are functionally enriched for endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) pathways. Moreover, rs28539249 locus was predicted to disrupt regulatory regions in human skeletal muscle with enriched epigenetic marks and binding affinity for CTCF. Supershift EMSA assays followed luciferase assays demonstrated the CTCF specifically binding to rs28539249-C allele leading to decreased transcriptional activity. Thus, the post-GWAS annotation confirmed the Asian-specific association of genetic variant in GPSM1 with T2DM, suggesting a role for the variant in the regulation in skeletal muscle.

A helitron-induced RabGDIα variant causes quantitative recessive resistance to maize rough dwarf disease.

Maize rough dwarf disease (MRDD), caused by various species of the genus Fijivirus, threatens maize production worldwide. We previously identified a quantitative locus qMrdd1 conferring recessive resistance to one causal species, rice black-streaked dwarf virus (RBSDV). Here, we show that Rab GDP dissociation inhibitor alpha (RabGDIα) is the host susceptibility factor for RBSDV. The viral P7-1 protein binds tightly to the exon-10 and C-terminal regions of RabGDIα to recruit it for viral infection. Insertion of a helitron transposon into RabGDIα intron 10 creates alternative splicing to replace the wild-type exon 10 with a helitron-derived exon 10. The resultant splicing variant RabGDIα-hel has difficulty being recruited by P7-1, thus leading to quantitative recessive resistance to MRDD. All naturally occurring resistance alleles may have arisen from a recent single helitron insertion event. These resistance alleles are valuable to improve maize resistance to MRDD and potentially to engineer RBSDV resistance in other crops.

Activator of G protein signaling 3 modulates prostate tumor development and progression.

Prostate cancer (PCa) is a leading cause of cancer death among men, with greater prevalence of the disease among the African American population in the USA. Activator of G-protein signaling 3 (AGS3/G-protein signaling modulator 1) was shown to be overexpressed in prostate adenocarcinoma relative to the prostate gland. In this study, we investigated the correlation between AGS3 overexpression and PCa malignancy. Immunoblotting analysis and real-time quantitative-PCR showed increase in AGS3 expression in the metastatic cell lines LNCaP (~3-fold), MDA PCa 2b (~2-fold), DU 145 (~2-fold) and TRAMP-C1 (~20-fold) but not in PC3 (~1-fold), relative to control RWPE-1. Overexpression of AGS3 in PC3, LNCaP and MDA PCa 2b enhanced tumor growth. AGS3 contains seven tetratricopeptide repeats (TPR) and four G-protein regulatory (GPR) motifs. Overexpression of the TPR or the GPR motifs in PC3 cells had no effect in tumor growth. Depletion of AGS3 in the TRAMP-C1 cells (TRAMP-C1-AGS3-/-) decreased cell proliferation and delayed wound healing and tumor growth in both C57BL/6 (~3-fold) and nude mice xenografts, relative to control TRAMP-C1 cells. TRAMP-C1-AGS3-/- tumors also exhibited a marked increase (~5-fold) in both extracellular signal-regulated kinase (ERK) 1/2 and P38 mitogen-activated protein kinase (MAPK) activation, which correlated with a significant increase (~3-fold) in androgen receptor (AR) expression, relative to TRAMP-C1 xenografts. Interestingly, overexpression of AGS3 in TRAMP-C1-AGS3-/- cells inhibited ERK activation and AR overexpression as compared with control TRAMP-C1 cells. Taken together, the data indicate that the effect of AGS3 in prostate cancer development and progression is probably mediated via a MAPK/AR-dependent pathway.

Recognition and stabilization of geranylgeranylated human Rab5 by the GDP Dissociation Inhibitor (GDI).

The small GTPase Rab5 is the key regulator of early endosomal fusion. It is post-translationally modified by covalent attachment of two geranylgeranyl (GG) chains to adjacent cysteine residues of the C-terminal hypervariable region (HVR). The GDP dissociation inhibitor (GDI) recognizes membrane-associated Rab5(GDP) and serves to release it into the cytoplasm where it is kept in a soluble state. A detailed new structural and dynamic model for human Rab5(GDP) recognition and binding with human GDI at the early endosome membrane and in its dissociated state is presented. In the cytoplasm, the GDI protein accommodates the GG chains in a transient hydrophobic binding pocket. In solution, two different binding modes of the isoprenoid chains inserted into the hydrophobic pocket of the Rab5(GDP):GDI complex can be identified. This equilibrium between the two states helps to stabilize the protein-protein complex in solution. Interprotein contacts between the Rab5 switch regions and characteristic patches of GDI residues from the Rab binding platform (RBP) and the C-terminus coordinating region (CCR) reveal insight on the formation of such a stable complex. GDI binding to membrane-anchored Rab5(GDP) is initially mediated by the solvent accessible switch regions of the Rab-specific RBP. Formation of the membrane-associated Rab5(GDP):GDI complex induces a GDI reorientation to establish additional interactions with the Rab5 HVR. These results allow to devise a detailed structural model for the process of extraction of GG-Rab5(GDP) by GDI from the membrane and the dissociation from targeting factors and effector proteins prior to GDI binding.

Role of G-proteins and phosphorylation in the distribution of AGS3 to cell puncta.

Activator of G-protein signaling 3 (AGS3, also known as GPSM1) exhibits broad functional diversity and oscillates among different subcellular compartments in a regulated manner. AGS3 consists of a tetratricopeptide repeat (TPR) domain and a G-protein regulatory (GPR) domain. Here, we tested the hypothesis that phosphorylation of the AGS3 GPR domain regulates its subcellular distribution and functionality. In contrast to the cortical and/or diffuse non-homogeneous distribution of wild-type (WT) AGS3, an AGS3 construct lacking all 24 potential phosphorylation sites in the GPR domain localized to cytosolic puncta. This change in localization was revealed to be dependent upon phosphorylation of a single threonine amino acid (T602). The punctate distribution of AGS3-T602A was rescued by co-expression of Gαi and Gαo but not Gαs or Gαq Following treatment with alkaline phosphatase, both AGS3-T602A and WT AGS3 exhibited a gel shift in SDS-PAGE as compared to untreated WT AGS3, consistent with a loss of protein phosphorylation. The punctate distribution of AGS3-T602A was lost in an AGS3-A602T conversion mutant, but was still present upon T602 mutation to glutamate or aspartate. These results implicate dynamic phosphorylation as a discrete mechanism to regulate the subcellular distribution of AGS3 and associated functionality.

A Residue outside the Binding Site Determines the Gα Binding Specificity of GoLoco Motifs.

GoLoco motif-containing proteins regulate the nucleotide-binding state of Gα proteins in various signaling pathways. As guanine nucleotide dissociation inhibitors (GDIs), they bind Gα·GDP and inhibit GDP to GTP exchange. GoLoco proteins show binding selectivity toward different members of the Gα family. Although the Gαi1·GDP/RGS14 crystal structure explains the specific binding selectivity of the RGS14 GoLoco domain well, the mechanism of selective binding has not been understood for the more general features of short GoLoco domains found in tandem arrays in proteins like GPSM2/LGN/ dPins and GPSM1/AGS3. We explored the mechanism of differential interactions of GoLoco protein LGN with hGαi3 and hGαo. By combining mutagenesis experiments and molecular dynamics simulations, we identified a residue (Asp229 in hGαi3) away from the binding interface that remarkably affects the interaction between LGN and hGαi/o. A negatively charged residue at this position is required for high binding affinity. This affinity regulation mechanism was further verified by the cases of hGαi2 and dGαo, suggesting that this pathway is conserved among members of the Gα family.