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Background: Interleukin-12 is a potent proinflammatory cytokine that proliferates and activates T cells, NK cells and differentiates Th1 cells. Translation of IL-12 for cancer treatment has been hindered by lethal toxicities due to cytokine release syndrome and there are currently no approved IL-12 therapies. To minimize the severe toxicities while maintaining potency, we have developed ON-BOARD, an ultra-pH sensitive nanoparticle platform for masked and targeted delivery of payloads to the acidic tumor microenvironment. The clinical feasibility of ON-BOARD has been demonstrated by high tumor specificity of pegsitacianine in multiple tumor types from the Phase I and II clinical trials. Herein we report encapsulation and masked delivery of IL-12 to tumor-bearing mice using ON-BOARD, demonstrating significantly improved tolerability, anti-tumor efficacy, and potential for clinical translation. Methods: A mouse IL-12 fused with Fc was formulated in ON-BOARD nanoparticles. Particle properties were characterized and lead formulations were identified by in vitro screening to determine pH-mediated bioactivity in reporter and ELISA assays and stability in mouse plasma. In vivo studies were performed to compare the activity of unencapsulated IL-12 to ON-BOARD/IL-12 formulations. PD response was evaluated by measuring systemic cytokine levels in plasma, while clinical chemistry was performed to evaluate liver and kidney functions. Anti-tumor efficacy of ON-BOARD/IL-12 formulations was performed in mice bearing syngeneic MC38 colorectal cancer tumors compared to unencapsulated IL-12. Results: ON-BOARD/IL-12 formulations showed high encapsulation efficiency (>85%) and drug loading up to 20% wt. in uniformly distributed stable particles (Dh100-fold activation window between the acid-activated and intact formulations. Following incubation in mouse plasma the lead ON-BOARD formulations showed stable IL-12 encapsulation by an ELISA assay. In vivo, ON-BOARD/IL-12 formulations demonstrated significantly improved tolerability compared to unencapsulated IL-12. When dosed at 5µg/dose compared to unencapsulated protein at 1 µg/dose, ON-BOARD/IL-12 demonstrated reduced body weight loss (1,000-fold reduction in plasma IFNγ level which is known to be directly induced by IL-12 signaling. ON-BOARD/IL-12 formulations also demonstrated strong anti-tumor efficacy in MC38 tumor-bearing animals with >95% TGI and complete responders. Conclusions: The ON-BOARD platform demonstrated potential for masking toxicity and facilitating tumor-specific delivery of IL-12 proteins for cancer therapy. Citation Format: Qingtai Su, Stephen Gutowski, Irina Kalashnikova, Austin Burcham, Bhargavi Allu, Zirong Chen, Zhichen Sun, Jinming Gao, Ruolan Han, Jason B. Miller, Tian Zhao. Encapsulation of IL-12 with an ultra pH-sensitive nanoparticle platform improves tolerability and promotes antitumor response in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB001.

Bispecific antibodies are an emerging class of therapeutics for immune-oncology applications. T cell engagers (TCEs) target tumor-associated antigens (TAA) to eradicate cancer cells. TCEs for solid tumors have demonstrated encouraging preclinical efficacy but development has been challenging with dose-limiting toxicities due to on-target/off-tumor effect. To overcome the issue, we have developed ON-BOARD, an ultra-pH sensitive nanoparticle platform for masked and targeted delivery of payloads to the acidic tumor microenvironment (TME). Herein we report efficacious masked delivery of a TCE to tumors in mice using ON-BOARD demonstrating significantly improved tolerability and potential for clinical translation. TCEs were encapsulated in ON-BOARD nanoparticles and characterized for particle properties. The formulations were assessed in vitro under neutral or acid-activated conditions in TDCC assays. In vivo studies were performed in mice bearing “immune desert” pancreatic cancer. ON-BOARD tumor localization was measured by fluorescence while unencapsulated TCE and ON-BOARD/TCE pharmacokinetics was evaluated. PD studies evaluated immune-phenotype changes in tumors and draining lymph nodes, and systemic cytokine levels. Efficacy studies were performed in tumor-bearing mice comparing unencapsulated TCE to ON-BOARD-TCE as monotherapy and in combination with anti-4-1BB agonist therapy. ON-BOARD encapsulated TCEs have high encapsulation efficiency (>88%) in uniformly distributed stable particles (Dh The ON-BOARD platform demonstrated potential as an effective tool for masked tumor-specific delivery and improved tolerability of bispecific TCEs for cancer therapy. Citation Format: Qingtai Su, Stephen Gutowski, Gaurav Bharadwaj, Austin Burcham, Bhargavi Allu, Irina Kalashnikova, Zirong Chen, Ruolan Han, Jason B. Miller, Tian Zhao. Improved tolerability and tumor specific delivery of a therapeutic bispecific T cell engager using a pH-sensitive nanoparticle platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 815.

Background: Though antibodies for cancer treatment have achieved clinical and commercial success over the past few decades, a large portion are limited by suboptimal efficacy and on-target off-tumor toxicity due to ubiquitous expression of their targets. A plethora of molecular engineering approaches have been introduced to restrict the activity of antibodies solely to tumor instead of healthy tissue with the goal of improving the therapeutic index, such as masking the binding sites of antibodies with inhibitory domains. However, sophisticated modification is usually required while the outcomes are often mediocre. Previously we developed an ultra-pH sensitive nanoparticle platform named ON-BOARD. The strength of this technology stems from its ability to preferentially release the payload specifically in the acidic tumor microenvironment while staying intact in normal tissue. The safety and feasibility of using such a platform have been demonstrated by successful delivery of fluorophore to tumors for imaging of multiple tumor types in Phase I and II clinical trials with Pegsitacianine (formerly “ONM-100”). Pegsitacianine has been shown to be generally well-tolerated with an infusion-related reaction as the most common adverse event in the clinical trials conducted to date. Based on the clinical results, we present the ON-BOARD platform herein as a potential universal and effective tool for tumor specific activation and delivery of therapeutic antibodies without the need for sophisticated antibody chemistry or engineering. Methods: Biosimilar monoclonal antibodies of atezolizumab, cetuximab, pembrolizumab, trastuzumab, and ipilimumab with the same variable region sequences as the original pharmaceutical drugs were used to demonstrate encapsulation by the ON-BOARD platform and pH-dependent activation. Encapsulated antibodies were purified using SEC and the encapsulation efficiencies were quantified by HPLC. Particle size and uniformity were studied by DLS. The formulations were accessed for bioactivity in vitro under neutral pH or acidified conditions using appropriate cell-based reporter assays. Results: ON-BOARD nanoparticles successfully encapsulated the antibodies mentioned above without additional modification of the original antibody. Encapsulation efficiency ranged from 50-100%. The formulations were characterized as uniformly distributed particles < 100nm in size with good stability. In vitro assessment by cell-based reporter assays demonstrated > 100-fold activation window between the acid-activated and intact formulations. The pH-dependent activation was further confirmed by affinity and binding assay. Conclusions: The ON-BOARD pH-sensitive nanoparticle platform demonstrated potential as an effective and universal tool for tumor specific activation and delivery of antibody-based therapeutics. Citation Format: Gaurav Bharadwaj, Qingtai Su, Stephen Gutowski, Curran Parpia, Ashley Campbell, Jason Miller, Tian Zhao. Encapsulating therapeutic antibodies for tumor specific activation and delivery using a clinically validated pH-sensitive nanoparticle platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1734.

Background Interleukin-2 (IL-2) is a potent immunotherapy for treatment of metastatic melanoma and renal cell cancers. However, the clinical application has been hindered by immunosuppressive stimulation and unfavorable pharmacological properties that can induce life-threatening toxicities.1 Although strategies including ‘no-alpha’ muteins have been developed to provide target specificity at the molecular level,2 little has been done to improve tumor specificity and accumulation at tissue level. We developed ONM-400, a novel IL-2 encapsulating pH-activated nanoparticle that targets metabolic acidosis of cancer to improve the therapeutic index of IL-2 therapy. During circulation, IL-2 activity is sequestered within the nanoparticles. Upon entering the tumor microenvironment, IL-2 release is precisely and instantly triggered by acidic tumor pH, resulting in the selective deposition of active IL-2 at the site of disease. Methods A tumor-agnostic pH-activated nanoparticle with pH responsiveness similar to ONM-100, a cancer imaging agent currently in a Phase 2 clinical trial,3 has been developed for cytokine delivery. IL-2 was encapsulated within the nanoparticle using a proprietary method to produce ONM-400 and the physical properties were characterized. Activity of IL-2 in ONM-400 was evaluated using a bioluminescent cell-based assay for both its encapsulated (inactive) state and activated format. Tumor accumulation and biodistribution following intravenous injection (I.V.) of ONM-400 were evaluated in mice bearing head and neck tumors using fluorescent imaging. In vivo antitumor efficacy of ONM-400 after I.V. injection was studied in MC38 colon cancer-bearing mice and compared with unencapsulated IL-2 at the same dose. Results Quantitative analysis shows high encapsulation efficiency and drug loading density of IL-2 in ONM-400. At neutral pH, IL-2 bioactivity is effectively sequestered in ONM-400 through encapsulation which avoids IL-2 toxicity in normal tissue. Upon acid-triggered release, IL-2 bioactivity is rescued without compromise compared to unencapsulated IL-2 control. Significantly higher tumor accumulation and lower renal elimination were observed with ONM-400 in biodistribution studies as compared to free IL-2 control suggesting an alteration of pharmacokinetics of IL-2 after encapsulation. ONM-400 induced strong antitumor efficacy as a monotherapy in MC38 colon cancer-bearing mice (figure 1). After ONM-400 treatment 60% of the animals showed complete tumor regression and remained tumor free 60 days. Following a secondary MC38 challenge, 5/6 animals resisted tumor growth. Conclusions Tumor acidosis-driven accumulation and activation of ONM-400 provide a high local concentration of IL-2 within tumors resulting in strong antitumor response as a monotherapy. Tumor metabolic targeting pH-activatable nanoparticles provides a novel strategy to deliver immunomodulators for cancer treatment. Ethics Approval All animal experiments were reviewed and approved, and performed in accordance with, by Pennsylvania State College of Medicine Institutional Animal Care and Use Committee under Animal Protocol Number: 47682. References Siegel JP, Puri RK. Interleukin-2 Toxicity. J Clin Oncol, 1991; 9: 694–704. Rosalia RA, Arenas-Ramirez N, Bouchaud G, Raeber, ME, Boyman O. Use of enhanced interleukin-2 formulations for improved immunotherapy against cancer. Curr Opin Chem Biol, 2014; 23: 39–46. Voskuil FJ, Steinkamp PJ, Zhao T, van der Vegt B, Koller M, Doff JJ, Jayalakshmi Y, Hartung JP, Gao, Sumer BD, Witjes MJH, van Dam GM, SHINE study group. Exploiting metabolic acidosis in solid cancers using a tumor-agnostic pH-activatable nanoprobe for fluorescence-guided surgery. Nat Commun 2020;11:3257.

Background: The stimulator of interferon genes (STING) plays a central role in innate immune response against infection and cancer. Naturally, the cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)-STING pathway adopts polyvalent interactions to form higher-order assemblies to achieve a specific and rapid response while limiting unnecessary stimulation by host DNA. Several cyclic di-nucleotide (CDN) and non-CDN small molecule STING agonists have been studied in clinical trials showing limited therapeutic efficacy. ONM-501, a dual-activating STING agonist employs PC7A, a synthetic polymer that induces polyvalent STING condensation and prolongs innate immune activation. Mechanistically, PC7A binds to STING through a non-competitive surface site that is distinct from the binding pocket of CDN and non-CDN small molecule STING agonists. ONM-501 encapsulates the endogenous STING agonist cGAMP with the PC7A micelles offering dual ‘burst' and ‘sustained' STING activation. Effectiveness of using ONM-501 for immunotherapy against solid tumors has been demonstrated. Methods: Polyvalent interaction between PC7A and STING was studied and characterized by several different methods including ITC, a FRET assay between fluorophore labeled STING and PC7A and Nile-Red assay. The binding valency was investigated using a series of PC7A polymers with an increasing number of repeating units. The PC7A-STING binding site was elucidated using STING mutants produced by site-directed mutagenesis. STING activation was evaluated by measuring Ifnb1 and Cxcl10 expression using RT-qPCR. STING activation by PC7A and ONM-501 was also investigated using freshly resected human tissue. ONM-501 antitumor efficacy was evaluated in 6 different murine syngeneic tumor models. Results: PC7A activates STING through a non-canonical biomolecular condensation. It binds to a non-competitive surface site on the α5 helix of STING which is different from the CDN binding pocket. This binding was also retained with cGAMP-resistant STING variants (e.g., R232H). The formation of STING-PC7A condensate and the downstream activation were dependent on polymer repeating units. ONM-501 achieves a synergistic, rapid and sustained (6-24h) STING activation in vivo compared to cGAMP which peaked at 6h. Injection of ONM-501 in fresh human tissue resulted in >100-fold increase in cytokine expression while free CDN only showed marginal effect. Antitumor efficacy was demonstrated in MC38, CT26, B16F10, 4T1, A20 and TC-1 models after ONM-501 treatment. Complete response was observed when combined with anti-PD1 checkpoint blockade therapy. Conclusions: PC7A polymer achieves prolonged innate immune activation by polyvalent STING condensation through a distinct binding site. ONM-501 combines endogenous cGAMP with PC7A that potentially offers a synergistic strategy in spatiotemporal orchestration of immune environment for a highly effective immunotherapy against cancer. Citation Format: Suxin Li, Min Luo, Zhaohui Wang, Qiang Feng, Jonathan Wilhelm, Xu Wang, Wei Li, Jian Wang, Qingtai Su, Gaurav Bharadwaj, Jason Miller, Katy Torres, Stephen Gutowski, Agnieszka Cholka, Yang-xin Fu, Tian Zhao, Baran Sumer, Hongtao Yu, Jinming Gao. ONM-501 ― A synthetic polyvalent STING agonist for cancer immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P049.

Rho family GTPases regulate a wide range of cellular processes. This includes cellular dynamics where three subfamilies, Rho, Rac, and Cdc42, are known to regulate cell shape and migration though coordinate action. Activation of Rho proteins largely depends on Rho Guanine nucleotide Exchange Factors (RhoGEFs) through a catalytic Dbl homology (DH) domain linked to a pleckstrin homology (PH) domain that subserves various functions. The PH domains from Lbc RhoGEFs, which specifically activate RhoA, have been shown to bind to activated RhoA. Here, p190RhoGEF, is shown to also bind Rac1•GTP. Crystal structures reveal that activated Rac1 and RhoA use their effector-binding surfaces to associate with the same hydrophobic surface on the PH domain. Both activated RhoA and Rac1 can stimulate exchange of nucleotide on RhoA by localization of p190RhoGEF to its substrate, RhoA•GDP, in vitro. The binding of activated RhoA provides a mechanism for positive feedback regulation as previously proposed for the family of Lbc RhoGEFs. In contrast, the novel interaction between activated Rac1 and p190RhoGEF reveals a potential mechanism for cross-talk regulation where Rac can directly effect stimulation of RhoA. The greater capacity of Rac1 to stimulate p190RhoGEF among the Lbc RhoGEFs suggests functional specialization.

Background: Immunotherapy has become a powerful strategy for cancer treatment. Immunomodulators such as cytokines (e.g. IL-2) and engineered antibody fragments (e.g. bispecific diabodies) can induce anti-tumor immune responses, but their clinical applications are limited by the unfavorable pharmacokinetic properties that can elicit serious dose-limiting toxicities (e.g. vascular leak syndrome). We have developed an ultra-pH sensitive nanoparticle platform, ONM-400, that delivers biomacromolecules to the acidic tumor microenvironment (TME) with high specificity. At normal physiological pH, the nanoparticles exist as intact micelles, stabilizing the payload, while at acidic tumor pH, they dissociate into unimers, releasing the payload. The feasibility of selectively targeting the acidic TME by this technology has been successfully validated by imaging of different types of tumors in a Phase 1 clinical trial. Using this platform, we have encapsulated and delivered IL-2 and an antibody fragment Fab to the TME to improve their pharmacokinetic properties. Methods: Multiple strategies have been exploited to encapsulate IL-2 and Fab antibody fragment including mixing, single emulsion, acid/base titration, double emulsion/solvent evaporation, and covalent conjugation. ONM-400•IL-2 and ONM-400•Fab complexes were purified by ultracentrifugation and characterized by dynamic light scattering. Encapsulation efficiency (EE) and drug loading (DL) were measured for each formulation using gel electrophoresis. In addition, IL-2 and Fab were labeled with a near-infrared dye (IRDye 800CW) to study the in vivo tumor accumulation and biodistribution after intravenous injection in mice bearing orthotopic head and neck tumors using a LI-COR Pearl camera. Results: ONM-400 nanoparticles were able to encapsulate IL-2 and Fab with high loading. In vivo imaging of ONM-400•IL-2 and ONM-400•Fab showed significantly higher tumor accumulation and less signal in the bladder compared to free IL-2 or Fab at the same dose after IV injection. ONM-400 formulations provided longer and increased exposure in tumor compared to the free biomacromolecules which decreased overtime. Quantitative analysis of the tumor signal at different time points indicated an alteration of the pharmacokinetics for IL-2 and Fab after encapsulation by ONM-400 nanoparticles. Biodistribution at 24h further revealed a lower accumulation in kidney and higher accumulation in tumor with ONM-400•IL-2 and ONM-400•Fab than free IL-2 or Fab respectively. Conclusion: The ONM-400 nanoparticles can efficiently encapsulate immunomodulators and alter their pharmacokinetics in vivo. These results suggest a great potential for using this ultra-pH sensitive nanoparticle technology to deliver immunomodulators to improve their efficacy and safety profile. Citation Format: Xinliang Ding, Jason Miller, Ashley Campbell, Jonathan Almazan, Stephen Gutowski, Tian Zhao. Delivery of immunomodulators to the acidic tumor microenvironment by ultra-pH sensitive nanoparticle technology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2867.

Background: The efficacy of cancer vaccines requires induction of cancer-specific cytotoxic T-lymphocytes (CTL) to effectively clear established tumors. Orchestration of antigen presentation, co-stimulatory signaling, and innate cytokine signals are necessary steps for tumor-specific T cell activation. The ONM-500 nanovaccine platform utilizes a novel pH-sensitive polymer that forms an antigen-encapsulating nanoparticle and functions both as a carrier for antigen delivery to dendritic cells and as an adjuvant, activating the stimulator of interferon genes (STING) pathway and generating a CD8+ CTL response. Peptide antigens have translational challenges due to limited stability, HLA-type-specific antigen sequence recognition and presentation. Full-length protein antigens alleviate HLA subtype limitation, allowing coverage of multi-immunogenic T cell epitopes in patients. Using the ONM-500 platform with the full-length recombinant E6/E7 oncoproteins from human papillomavirus (HPV) cancers, we have demonstrated great antitumor efficacy in an HPV positive cancer model in animals. Methods: Based on the previously demonstrated STING-dependent T cell activation by ONM-500, the nanovaccine was formulated with full-length HPV16 E6/E7 proteins, and the nanoparticle properties and antigen loading were characterized. In vivo lymph node accumulation following subcutaneous administration was evaluated using fluorophore-labeled ONM-500. Direct binding of ONM-500 polymer to human STING was evaluated using a pull-down assay. Antitumor efficacy of ONM-500 nanovaccine with full length E6/E7 was evaluated using a TC-1 model which overexpresses HPV16 E6/E7 and compared with ONM-500 formulated with peptide antigen. Long-term anti-tumor memory was evaluated in a rechallenge study after 60 days in tumor-free animals. Results: Characterization of ONM-500 nanovaccine shows reproducible particle chemical and physical properties and antigen loading. The nanoparticle size substantiates the effective lymph node accumulation for antigen cross-presentation in dendritic cells following subcutaneous administration. The pull-down assay showed effective binding of the ONM-500 polymer to STING. In TC-1 tumors, ONM-500 nanovaccine containing full-length E6/E7 protein showed 100% overall survival at 55 days. Tumor growth inhibition was also improved over peptide antigen formulated ONM-500. A rechallenge study demonstrated long-term antigen-specific anti-tumor memory response. Conclusions: ONM-500 STING-activating nanovaccines effectively deliver antigens in vivo to lymph nodes to elicit an antigen-specific CTL response. The anti-tumor efficacy in the animal tumor model demonstrates the potential of ONM-500 as a general STING-activating cancer vaccine platform, and full-length E6/E7 incorporated ONM-500 is being developed for HPV-associated cancers. Citation Format: Jason Miller, Min Luo, Hua Wang, Zhaohui Wang, Xinliang Ding, Ashley Campbell, Jonathan Almazan, Stephen Gutowski, Zhijian Chen, Jinming Gao, Tian Zhao. ONM-500: A STING-activating therapeutic nanovaccine platform for cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4577.

PDZ domains are abundant protein interaction modules and typically recognize a short motif at the C terminus of their ligands, with a few residues in the motif endowing the binding specificity. The sequence-based rules, however, cannot fully account for the specificity between the vast number of PDZ domains and ligands in the cell. Plexins are transmembrane receptors that regulate processes such as axon guidance and angiogenesis. Two related guanine nucleotide exchange factors (GEFs), PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), use their PDZ domains to bind class B plexins and play critical roles in signaling. Here, we present the crystal structure of the full-length cytoplasmic region of PlexinB2 in complex with the PDZ domain of PDZ-RhoGEF. The structure reveals that, in addition to the canonical C-terminal motif/PDZ interaction, the 3D domain of PlexinB2 forms a secondary interface with the PDZ domain. Our biophysical and cell-based assays show that the secondary interface contributes to the specific interaction between plexin and PDZ-RhoGEF and to signaling by plexin in the cell. Formation of secondary interfaces may be a general mechanism for increasing affinity and specificity of modular domain-mediated interactions.

The monomeric Rho GTPases are essential for cellular regulation including cell architecture and movement. A direct mechanism for hormonal regulation of the RhoA-type GTPases is their modulation by the G12 and G13 proteins via RH (RGS homology) containing RhoGEFs. In addition to the interaction of the G protein α subunits with the RH domain, activated RhoA also binds to the pleckstrin homology (PH) domain of PDZRhoGEF. The latter interaction is now extended to all seven members of the homologous Lbc family of RhoGEFs which includes the RH-RhoGEFs. This is evinced by direct measurements of binding or through effects on selected signaling pathways in cells. Overexpression of these PH domains alone can block RhoA-dependent signaling in cells to various extents. Whereas activated RhoA does not modulate the intrinsic activity of the RhoGEFs, activated RhoA associated with phospholipid vesicles can facilitate increased activity of soluble RhoGEFs on vesicle-delimited substrate (RhoA-GDP). This demonstrates feasibility of the hypothesis that binding of activated RhoA to the PH domains acts as a positive feedback mechanism. This is supported by cellular studies in which mutation of this binding site on PH strongly attenuates the stimulation of RhoA observed by overexpression of five of the RhoGEF DH-PH domains. This mutation is even more dramatic in the context of full-length p115RhoGEF. The utilization of this mechanism by multiple RhoGEFs suggests that this regulatory paradigm may be a common feature in the broader family of RhoGEFs.

The regulator of G protein signaling homology (RH) Rho guanine nucleotide exchange factors (RhoGEFs) (p115RhoGEF, leukemia-associated RhoGEF, and PDZ-RhoGEF) contain an RH domain and are specific GEFs for the monomeric GTPase RhoA. The RH domains interact specifically with the α subunits of G12 heterotrimeric GTPases. Activated Gα13 modestly stimulates the exchange activity of both p115RhoGEF and leukemia-associated RhoGEF but not PDZ-RhoGEF. Because all three RH-RhoGEFs can localize to the plasma membrane upon expression of activated Gα13, cellular localization of these RhoGEFs has been proposed as a mechanism for controlling their activity. We use a small molecule-regulated heterodimerization system to rapidly control the localization of RH-RhoGEFs. Acute localization of the proteins to the plasma membrane activates RhoA within minutes and to levels that are comparable with activation of RhoA by hormonal stimulation of G protein-coupled receptors. The catalytic activity of membrane-localized RhoGEFs is not dependent on activated Gα13. We further show that the conserved RH domains can rewire two different RacGEFs to activate Rac1 in response to a traditional activator of RhoA. Thus, RH domains act as independent detectors for activated Gα13 and are sufficient to modulate the activity of RhoGEFs by hormones via mediating their localization to substrate, membrane-associated RhoA.

Rho GTPases play a fundamental role in numerous cellular processes that are initiated by extracellular stimuli including agonists that work through G protein-coupled receptors. A direct pathway for such regulation was elucidated by the identification of p115 RhoGEF, an exchange factor for RhoA that is activated through its RGS domain by G alpha(13). Endogenous p115 RhoGEF was found mainly in the cytosol of serum-starved cells but partially localized to membranes in cells stimulated with lysophosphatidic acid. Overexpressed p115 RhoGEF was equally distributed between membranes and cytosol; either the RGS or pleckstrin homology domain was sufficient for this partial targeting to membranes. Removal of the pleckstrin homology domain dramatically reduced the in vitro rate of p115 RhoGEF exchange activity. Deletion of amino acids 252--288 in the linker region between the RGS domain and the Dbl homology domain or of the last 150 C-terminal amino acids resulted in non-additive reduction of in vitro exchange activity. In contrast, p115 RhoGEF pieces lacking this extended C terminus were over 5-fold more active than the full-length exchange factor in vivo. These results suggest that p115 RhoGEF is inhibited in the cellular milieu through modification or interaction of inhibitory factors with its C terminus. Endogenous p115 RhoGEF that was immunoprecipitated from cells stimulated with lysophosphatidic acid or sphingosine 1-phosphate was more active than when the enzyme was immunoprecipitated from untreated cells. This indicates an additional and potentially novel long lived mechanism for regulation of p115 RhoGEF by G protein-coupled receptors.

RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G(12) class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated α subunits of G(12) and G(13). Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by Gα(13), the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, we identify an additional binding site for activated Gα(13) in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of Gα(13) docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the α3b helix of DH reduces binding to activated Gα(13) and ablates the stimulation of p115 by Gα(13). Complementary mutations at the predicted DH-binding site in the αB-αC loop of the helical domain of Gα(13) also affect stimulation of p115 by Gα(13). Although the GAP activity of p115 is not required for stimulation by Gα(13), two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of Gα(13) to the RH domain facilitates direct association of Gα(13) to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling.

In neuroblastoma-glioma hybrid cells, bradykinin has dual modulatory effects on ion channels: it activates a K+ current as well as inhibits the voltage-dependent Ca2+ current (ICa,V). Both of these actions are mediated by pertussis toxin-insensitive G proteins. Antibodies raised against the homologous Gq and G11 proteins suppress only the activation of the K+ current; this suggested that at least two distinct G protein pathways transduce diverse effects of this transmitter. Here, we show that the inhibition of ICa,V by bradykinin is suppressed selectively by intracellular application of antibodies specific for G13. This novel G protein may play a general role in the inhibition of ICa,V by pathways resistant to pertussis toxin.

The hydrolysis of phosphatidylcholine by phospholipase D (PLD) results in the production of phosphatidic acid and choline. An assay that uses an exogenous substrate was developed to measure this activity in membranes and solubilized preparations from HL60 cells. A cytosolic factor markedly enhanced PLD activity in membranes and was essential for GTP gamma S-dependent stimulation of an enriched preparation of PLD. The factor was purified to homogeneity from bovine brain cytosol and identified as a member of the ADP-Ribosylation Factor (ARF) subfamily of small G proteins. Subsequently, recombinant myristoylated ARF1 was found to be a better activator of PLD activity than was the nonmyristoylated form. ARF proteins have been implicated recently as factors for regulation of intracellular vesicle traffic. The current finding suggests that PLD activity plays a prominent role in the action of ARF and that ARF may be a key component in the generation of second messengers via phospholipase D.

The subfamily of guanine nucleotide-binding regulatory (G proteins) designated Gq has been shown to regulate the activity of phospholipase C by reconstitution. However, the role of these proteins in hormonal regulation of this activity has not been demonstrated. Two antisera were used in attempts to interrupt this pathway. Antiserum W082, developed against a peptide representing an internal sequence in alpha q, was specific for alpha q by immunoblots but did not recognize the native protein. Antiserum X384 was developed against a peptide representing the 12 amino acids of the common carboxyl termini of alpha q and alpha 11. It had a broader specificity for this subfamily of G protein alpha subunits and recognized the native proteins. Antiserum X384 specifically immunoprecipitated alpha q and its homologs from purified preparations and detergent extracts of membranes. Affinity-purified antibodies attenuated stimulation of phosphatidylinositide 4,5-bisphosphate hydrolysis by bradykinin, angiotensin, and histamine in membranes derived from NG108-15 cells, rat liver, and 1321N1 cells, respectively. Activation of the phospholipase C activity by guanosine 5'-3-O-(thio)triphosphate alone was also inhibited. Inclusion of the peptide to which the antisera were raised blocked the effect of the antibody. In contrast, affinity-purified W082, which did not recognize native proteins, did not alter regulation of phospholipase C. This indicates that the Gq family of signaling proteins can couple to several receptors and is responsible for the hormonal regulation of phospholipase C in these diverse systems. The further generality of this regulatory pathway remains to be established.

MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that can regulate the c-Jun amino-terminal kinase (JNK) MAP kinase cascade. MEKK1 is comprised of a kinase domain and a long amino-terminal regulatory domain. This amino-terminal domain has a scaffold function in that it can assemble modules of the JNK and ERK MAP kinase cascades. Recently, we have demonstrated that MEKK1 binds to p115 Rho GTPase-activating protein, which has GTPase-activating protein activity toward RhoA. Thus, we tested whether Rho GTPases interact with the regulatory domain of MEKK1. RhoA, but not Rac or Cdc42, binds to a site in the aminoterminal one-third of MEKK1, which includes its PHD domain. The interaction is prevented by mutation of the essential cysteine in the MEKK1 PHD domain. Rho-GTP stimulates the kinase activity of full-length MEKK1 as much as 10-fold toward MEK4 but does not appear to be ubiquitinated by MEKK1 under conditions that result in modification of ERK2. In summary, we have characterized a novel point at which Rho GTPases impinge upon the regulation and function of MEKK1.

The Rho family of monomeric GTPases plays a prominent role in the regulation of cell shape and movement. The activity of these proteins is dependent on a nucleotide cycle that is regulated by a combination of guanine nucleotide exchange factors (GEFs) that facilitate exchange of GTP for GDP on the Rho proteins, and GTPase-activating proteins (GAPs) that stimulate hydrolysis of GTP bound to Rho. The GEFs promote activation, that is, formation of the GTP-bound state, whereas proteins with GAP function serve to inactivate the GTPases. The heterotrimeric G proteins that mediate regulation by a variety of extracellular stimuli are also controlled by a GDP/GTP cycle. Integral membrane receptors for detection of hormones and other stimuli act as the GEFs to provide stimulatory inputs and a family of RGS (regulator of G protein signaling) proteins as GAPs that can effect either inhibitory or downstream regulation. The GEFs for the Rho family of GTPases compose a large and growing family of proteins characterized by conserved, tandem DH (Dbl homology) and PH (pleckstrin homology) domains. These exchange factors stimulate Rho proteins with various selectivities for three defined functional groups represented by RhoA, Racl, and Cdc42. p115 RhoGEF has been purified and cloned as a GEF that is selective for Rho. The subsequent identification of an RGS domain in the N terminus of p115, which has specificity for the G12 family of heterotrimeric G proteins, and the regulation of GEF activity by the activated Ga∼3 subunit, defines this protein as a direct link for coupling regulation between these two G protein pathways.

Phospholipase D (PLD) hydrolyzes phospholipids into phosphatidic acid (PA) and the base head groups. In mammalian cells, this activity plays an active role in signal transduction by acting as a mediator for a variety of extracellular stimuli. Two mammalian PLD genes have been identified and the encoded enzymes expressed and characterized. The activity of PLD1 is regulated by the ADPribosylation factor (ARF) and Rho families of monomeric GTPases, classic isoforms of protein kinase C (PKC), and the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ). PLD2, although highly sensitive to PIP 2 has shown only modest response to ARF and appears unresponsive to Rho and PKC, in vitro . Characterization of PLD activity and identification of various regulatory molecules were greatly facilitated by the establishment of an assay for in vitro studies. Alternative methods for the assay of PLD described in this chapter facilitate measurement of basal activity and regulation in the absence of PIP 2 .

The heterotrimeric G proteins, G(12) and G(13), mediate signaling between G protein-coupled receptors and the monomeric GTPase, RhoA. One pathway for this modulation is direct stimulation by Galpha(13) of p115 RhoGEF, an exchange factor for RhoA. The GTPase activity of both Galpha(12) and Galpha(13) is increased by the N terminus of p115 Rho guanine nucleotide exchange factor (GEF). This region has weak homology to the RGS box sequence of the classic regulators of G protein signaling (RGS), which act as GTPase-activating proteins (GAP) for G(i) and G(q). Here, the RGS region of p115 RhoGEF is shown to be distinctly different in that sequences flanking the predicted "RGS box" region are required for both stable expression and GAP activity. Deletions in the N terminus of the protein eliminate GAP activity but retain substantial binding to Galpha(13) and activation of RhoA exchange activity by Galpha(13). In contrast, GTRAP48, a homolog of p115 RhoGEF, bound to Galpha(13) but was not stimulated by the alpha subunit and had very poor GAP activity. Besides binding to the N-terminal RGS region, Galpha(13) also bound to a truncated protein consisting only of the Dbl homology (DH) and pleckstrin homology (PH) domains. However, Galpha(13) did not stimulate the exchange activity of this truncated protein. A chimeric protein, which contained the RGS region of GTRAP48 in place of the endogenous N terminus of p115 RhoGEF, was activated by Galpha(13). These results suggest a mechanism for activation of the nucleotide exchange activity of p115 RhoGEF that involves direct and coordinate interaction of Galpha(13) to both its RGS and DH domains.

The activity of phospholipase D (PLD) is regulated by a variety of hormonal stimuli and provides a mechanistic pathway for response of cells to extracellular stimuli. The two identified mammalian PLD enzymes possess highly homologous C termini, which are required for catalytic activity. Mutational analysis of PLD1 and PLD2 reveals that modification of as little as the C-terminal threonine or the addition of a single alanine attenuates activity of the enzyme. Protein folding appears to be intact because mutant enzymes express to similar levels in Sf9 cells and addition of peptides representing the C-terminal amino acids, including the simple hexamer PMEVWT, restores partial activity to several of the mutants. Analysis of several mutants suggests a requirement for the hydrophobic reside at the -2-position but not an absolute requirement for the hydroxyl side chain of threonine at the C terminus. The inability of peptides amidated at their C termini to effect restoration of activity indicates the involvement of the C-terminal alpha carboxyl group in functional activity of these enzymes. The ability of peptides to restore activity to PLD enzymes mutated at the C terminus suggests a flexible interaction of this portion of the molecule with a catalytic core constructed on conserved HKD motifs. Participation of these C termini residues in either stabilization of the catalytic site or the enzymatic reaction itself remains to be determined. This requirement for the C terminus provides an excellent potential site for interaction with regulatory proteins that may either enhance or down-regulate the activity of these enzymes in vitro.

Phospholipase D (PLD) activity from membranes of cultured cells can be activated by guanosine 5'-O-(3-thiotriphosphate) and the small GTP-dependent protein, Arf. While this activity was readily apparent in membranes from HL60 cells, it was much lower or not observable in membranes from various mammalian tissues. However, extraction of porcine brain membranes with detergent and subsequent chromatography with SP-Sepharose revealed a large peak of Arf-sensitive PLD activity. This activity has been enriched through several steps of chromatography and characterized with respect to size, nucleotide specificity, and sensitivity to different Arf and Arf-like proteins. Hydrodynamic analysis indicated that the enriched PLD had an s20,w of 5.1 and a Stokes radius of 4.3 nm. These parameters indicate that the enzyme has an apparent molecular mass of 95,000 Da. Effective stimulation of the enriched enzyme was achieved with GTP as well as nonhydrolyzable analogs. All of the Arf subtypes tested were effective activators of PLD activity. Arf derived from yeast could activate mammalian PLD but with lower potency. The Arf-related Arl proteins were ineffective. PLD that has been highly enriched retained a requirement for phosphatidylinositol 4,5-bisphosphate for efficient expression of activity. Additionally, the ability of recombinant or purified porcine brain Arf to stimulate PLD activity was reduced relative to impure fractions of Arf activity. Thus, porcine PLD that has been purified about 5,000-10,000-fold is synergistically activated by Arf in combination with other cytosolic components that are described in the accompanying paper (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950). Taken together, these data suggest that physiological regulation of Arf-sensitive PLD may involve the coordinate assembly of several interacting regulatory subunits.

In NG 108-15 cells, bradykinin (BK) activates a potassium current (I K,BK ) and inhibits the voltage-dependent calcium current (I Ca,V ). BK also stimulates a phosphatidylinositol-specific phospholipase C (PI-PLC). The subsequent release of inositol 1,4,5-trisphosphate and increase in intracellular calcium contribute to I K,BK , through activation of a calcium-dependent potassium current. In membranes from these cells, stimulation of PI-PLC by BK is mediated by G q and/or G 11 two homologous, pertussis toxin-insensitive G proteins. Here, we have investigated the role of G q/l1 in the electrical responses to BK. GTPγS mimicked and occluded both actions of BK, and both effects were insensitive to pertussis toxin. Perfusion of an anti-G q/11α antibody into the pipette suppressed I K,BK , but not the inhibition of I Ca,V by BK. Thus, BK couples to I K,BK via G q/11 , but coupling to I Ca,V is most likely via a different, pertussis toxin-insensitive G protein.

Heterotrimeric GTP-dependent regulatory proteins (G-proteins) mediate modulation by many cell surface receptors. Activation of the G-proteins promotes dissociation of their α and βγ subunits. The similarity of behaviour of βγ subunits derived from a variety of G-proteins has led to their use as affinity reagents for the analysis of the more unique a subunits. The evolution of these uses is presented. One of the more insightful results was the isolation of a new class of G -protein α subunits (the αqsubfamily) which have been shown to regulate the activity of a phospholipase C (PLC) specific for phosphatidylinositols. The experimental evidence leading to this conclusion is discussed. The activation by αqincreases the apparentVmaxof the β isoform of phosphatidylinositol-specific phospholipase C (PLCβ) and can be modulated by βγ subunits.

A small subset of cellular proteins are covalently modified by the addition of isoprenoid groups. These include p21ras, fungal mating factors, and nuclear lamins, which are isoprenylated at carboxyl-terminal cysteine residues with a 15-carbon farnesyl group. The similarity of the carboxyl-terminal sequences of these proteins with the alpha and gamma subunits of signal-transducing guanine nucleotide-binding regulatory proteins (G proteins) prompted examination of isoprenylation of G protein subunits. PC-12 cells were incubated with the isoprenoid precursor [3H]mevalonolactone. The beta and gamma subunits were isolated by specific association with an affinity column of immobilized alpha subunits. The gamma subunits were radiolabeled, and the tritiated lipid released from them by treatment with methyl iodide comigrated chromatographically with the 20-carbon isoprenoid geranylgeraniol. Label was not detected in G protein alpha or beta subunits. Isoprenylation of gamma subunits by the geranylgeranyl group is presumed to contribute to the association of G proteins with membranes.