Single-Cell Analyses Reveal Megakaryocyte-Biased Hematopoiesis in Myelofibrosis and Identify Mutant Clone-Specific Targets.

Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34+ lineage- hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.

Chitinase 3-like-1 Inhibits Innate Antitumor and Tissue Remodeling Immune Responses by Regulating CD47-SIRPα- and CD24-Siglec10-Mediated Phagocytosis.

Innate immune responses such as phagocytosis are critically linked to the generation of adaptive immune responses against the neoantigens in cancer and the efferocytosis that is essential for homeostasis in diseases characterized by lung injury, inflammation, and remodeling as in chronic obstructive pulmonary disease (COPD). Chitinase 3-like-1 (CHI3L1) is induced in many cancers where it inhibits adaptive immune responses by stimulating immune checkpoint molecules (ICPs) and portends a poor prognosis. CHI3L1 is also induced in COPD where it regulates epithelial cell death. In this study, we demonstrate that pulmonary melanoma metastasis inhibits macrophage phagocytosis by stimulating the CD47-SIRPα and CD24-Siglec10 phagocytosis checkpoint pathways while inhibiting macrophage "eat me" signals from calreticulin and HMGB1. We also demonstrate that these effects on macrophage phagocytosis are associated with CHI3L1 stimulation of the SHP-1 and SHP-2 phosphatases and inhibition of the accumulation and phosphorylation of cytoskeleton-regulating nonmuscle myosin IIa. This inhibition of innate immune responses such as phagocytosis provides a mechanistic explanation for the ability of CHI3L1 to stimulate ICPs and inhibit adaptive immune responses in cancer and diseases such as COPD. The ability of CHI3L1 to simultaneously inhibit innate immune responses, stimulate ICPs, inhibit T cell costimulation, and regulate a number of other oncogenic and inflammation pathways suggests that CHI3L1-targeted therapeutics are promising interventions in cancer, COPD, and other disorders.

Targeted volume correlative light and electron microscopy of an environmental marine microorganism.

Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional (3D) ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods along with the complexity and heterogeneity of environmental samples pose great technical challenges. In the present study, we used a workflow based on a specific electron microscopy sample preparation method compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate, which we identified as Ensiculifera tyrrhenica, with quantitative characterization of multiple organelles. We show that this cell contains a single convoluted chloroplast and show the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed partial chromatin unfolding, potentially associated with transcription activity in these organisms, in which chromosomes are permanently condensed. Together with providing insights in dinoflagellate biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.

Dynamic subcellular translocation of V-type H+ -ATPase is essential for biomineralization of the diatom silica cell wall.

Diatom cell walls, called frustules, are main sources of biogenic silica in the ocean and their intricate morphology is an inspiration for nanoengineering. Here we show dynamic aspects of frustule biosynthesis involving acidification of the silica deposition vesicle (SDV) by V-type H+  ATPase (VHA). Transgenic Thalassiosira pseudonana expressing the VHA B subunit tagged with enhanced green fluorescent protein (VHAB -eGFP) enabled subcellular protein localization in live cells. In exponentially growing cultures, VHAB -eGFP was present in various subcellular localizations including the cytoplasm, SDVs and vacuoles. We studied the role of VHA during frustule biosynthesis in synchronized cell cultures of T. pseudonana. During the making of new biosilica components, VHAB -eGFP first localized in the girdle band SDVs, and subsequently in valve SDVs. In single cell time-lapse imaging experiments, VHAB -eGFP localization in SDVs precluded accumulation of the acidotropic silica biomineralization marker PDMPO. Furthermore, pharmacological VHA inhibition prevented PDMPO accumulation in the SDV, frustule biosynthesis and cell division, as well as insertion of the silicalemma-associated protein SAP1 into the SDVs. Finally, partial inhibition of VHA activity affected the nanoscale morphology of the valve. Altogether, these results indicate that VHA is essential for frustule biosynthesis by acidifying the SDVs and regulating the insertion of other structural proteins into the SDV.

Higher social cue utilisation improves communication, reduces perceived workload, and improves performance amongst ad hoc dyads in simulated rail control.

The objective was to examine whether ad hoc dyads with different collective social cue utilisation would record differences in performance and perceptions of workload during a simulated rail control task that incorporated distinct levels of demand. The frequency of two types of communicative statements was also examined as mediating factors: closing the loop and informative responding. A quasi-experimental design was employed using 40 dyadic teams. The results indicated that ad hoc teams whose members comprised higher social cue utilisation recorded relatively faster response times and perceived lower levels of workload, and engaged in a greater frequency of communicative statements that involved 'closing the loop' and 'informative responses'. Social cue utilisation also exerted an indirect effect on perceived workload through informative responding. The outcomes have theoretical implications for models of ad hoc team performance, and practical implications for the selection and training of teams that operate on an ad hoc basis. Practitioner summary: This study indicates that, in the context of simulation tasks requiring teamwork, higher social cue utilisation amongst ad hoc team members is associated with communication, workload perception, and overall performance.

MicroRNA-155 induction via TNF-α and IFN-γ suppresses expression of programmed death ligand-1 (PD-L1) in human primary cells.

Programmed death ligand-1 (PD-L1) is a critical regulator of T cell function contributing to peripheral immune tolerance. Although it has been shown that posttranscriptional regulatory mechanisms control PD-L1 expression in cancer, it remains unknown whether such regulatory loops operate also in non-transformed cells. Here we studied PD-L1 expression in human dermal lymphatic endothelial cells (HDLECs), which play key roles in immunity and cancer. Treatment of HDLECs with the pro-inflammatory cytokines IFN-γ and TNF-α synergistically up-regulated PD-L1 expression. IFN-γ and TNF-α also affected expression of several microRNAs (miRNAs) that have the potential to suppress PD-L1 expression. The most highly up-regulated miRNA following IFN-γ and TNF-α treatment in HDLECs was miR-155, which has a central role in the immune system and cancer. Induction of miR-155 was driven by TNF-α, the effect of which was significantly enhanced by IFN-γ. The PD-L1 3'-UTR contains two functional miR-155-binding sites. Endogenous miR-155 controlled the kinetics and maximal levels of PD-L1 induction upon IFN-γ and TNF-α treatments. We obtained similar findings in dermal fibroblasts, demonstrating that the IFN-γ/TNF-α/miR-155/PD-L1 pathway is not restricted to HDLECs. These results reveal miR-155 as a critical component of an inflammation-induced regulatory loop controlling PD-L1 expression in primary cells.

The Role of Social Cue Utilization and Closing-the-Loop Communication in the Performance of Ad Hoc Dyads.

OBJECTIVE: To examine whether social cue utilization impacts the performance of ad hoc dyads through its relationship with closing the loop, a communication process whereby team members respond more frequently to initiating statements made by others. BACKGROUND: There lacks unequivocal experimental evidence for any single cognitive-based process that might predict the performance of ad hoc teams. METHOD: Using a quasi-experimental design, 80 participants were classified into 40 dyads based on their levels of social cue utilization and attempted a team problem-solving task. A serial mediation model revealed an indirect effect of social cue utilization on the performance of ad hoc dyads through closing the loop. RESULTS: Analyses indicated that social cue utilization impacts on the performance of ad hoc dyads independently of nonverbal reasoning ability and emotional intelligence. Further, the level of social cue utilization within dyads exhibits a positive indirect impact on the performance of ad hoc dyads through closing the loop. CONCLUSION: Ad hoc dyads with higher levels of social cue utilization engaged in a greater frequency of closing-the-loop statements and showed better subsequent performance on a problem-solving task in comparison to dyads with lower levels of social cue utilization. APPLICATION: Potential applications include the optimization of ad hoc team composition within high reliability environments like aviation and power control as well as improving training interventions with a specific mechanism for improving the performance of ad hoc teams.

The role of the hypothalamic paraventricular nucleus and the organum vasculosum lateral terminalis in the control of sodium appetite in male rats.

Angiotensin II (AngII) and aldosterone cooperate centrally to produce a robust sodium appetite. The intracellular signaling and circuitry that underlie this interaction remain unspecified. Male rats pretreated with both deoxycorticosterone (DOC; a synthetic precursor of aldosterone) and central AngII exhibited a marked sodium intake, as classically described. Disruption of inositol trisphosphate signaling, but not extracellular-regulated receptor kinase 1 and 2 signaling, prevented the cooperativity of DOC and AngII on sodium intake. The pattern of expression of the immediate early gene product cFos was used to identify key brain regions that may underlie this behavior. In the paraventricular nuclei (PVN) of the hypothalamus, DOC pretreatment diminished both AngII-induced cFos induction and neurosecretion of oxytocin, a peptide expressed in the PVN. Conversely, in the organum vasculosum lateral terminalis (OVLT), DOC pretreatment augmented cFos expression. Immunohistochemistry identified a substantial presence of oxytocin fibers in the OVLT. In addition, when action potentials in the PVN were inhibited with intraparenchymal lidocaine, AngII-induced sodium ingestion was exaggerated. Intriguingly, this treatment also increased the number of neurons in the OVLT expressing AngII-induced cFos. Collectively, these results suggest that the behavioral cooperativity between DOC and AngII involves the alleviation of an inhibitory oxytocin signal, possibly relayed directly from the PVN to the OVLT.

Mechanism of erythropoietin regulation by angiotensin II.

Erythropoietin (EPO) is the primary regulator of red blood cell development. Although hypoxic regulation of EPO has been extensively studied, the mechanism(s) for basal regulation of EPO are not well understood. In vivo studies in healthy human volunteers and animal models indicated that angiotensin II (Ang II) and angiotensin converting enzyme inhibitors regulated blood EPO levels. In the current study, we found that Ang II induced EPO expression in situ in murine kidney slices and in 786-O kidney cells in culture as determined by reverse transcription polymerase chain reaction. We further investigated the signaling mechanism of Ang II regulation of EPO in 786-O cells. Pharmacological inhibitors of Ang II type 1 receptor (AT1R) and extracellular signal-regulated kinase 1/2 (ERK1/2) suppressed Ang II transcriptional activation of EPO. Inhibitors of AT2R or Src homology 2 domain-containing tyrosine phosphatase had no effect. Coimmunoprecipiation experiments demonstrated that p21Ras was constitutively bound to the AT1R; this association was increased by Ang II but was reduced by the AT1R inhibitor telmisartan. Transmembrane domain (TM) 2 of AT1R is important for G protein-dependent ERK1/2 activation, and mutant D74E in TM2 blocked Ang II activation of ERK1/2. Ang II signaling induced the nuclear translocation of the Egr-1 transcription factor, and overexpression of dominant-negative Egr-1 blocked EPO promoter activation by Ang II. These data identify a novel pathway for basal regulation of EPO via AT1R-mediated Egr-1 activation by p21Ras-mitogen-activated protein kinase/ERK kinase-ERK1/2. Our current data suggest that Ang II, in addition to regulating blood volume and pressure, may be a master regulator of erythropoiesis.

Comparative genomics of transport proteins in developmental bacteria: Myxococcus xanthus and Streptomyces coelicolor.

BACKGROUND: Two of the largest fully sequenced prokaryotic genomes are those of the actinobacterium, Streptomyces coelicolor (Sco), and the δ-proteobacterium, Myxococcus xanthus (Mxa), both differentiating, sporulating, antibiotic producing, soil microbes. Although the genomes of Sco and Mxa are the same size (~9 Mbp), Sco has 10% more genes that are on average 10% smaller than those in Mxa. RESULTS: Surprisingly, Sco has 93% more identifiable transport proteins than Mxa. This is because Sco has amplified several specific types of its transport protein genes, while Mxa has done so to a much lesser extent. Amplification is substrate- and family-specific. For example, Sco but not Mxa has amplified its voltage-gated ion channels but not its aquaporins and mechano-sensitive channels. Sco but not Mxa has also amplified drug efflux pumps of the DHA2 Family of the Major Facilitator Superfamily (MFS) (49 versus 6), amino acid transporters of the APC Family (17 versus 2), ABC-type sugar transport proteins (85 versus 6), and organic anion transporters of several families. Sco has not amplified most other types of transporters. Mxa has selectively amplified one family of macrolid exporters relative to Sco (16 versus 1), consistent with the observation that Mxa makes more macrolids than does Sco. CONCLUSIONS: Except for electron transport carriers, there is a poor correlation between the types of transporters found in these two organisms, suggesting that their solutions to differentiative and metabolic needs evolved independently. A number of unexpected and surprising observations are presented, and predictions are made regarding the physiological functions of recognizable transporters as well as the existence of yet to be discovered transport systems in these two important model organisms and their relatives. The results provide insight into the evolutionary processes by which two dissimilar prokaryotes evolved complexity, particularly through selective chromosomal gene amplification.

Tailoring confocal microscopy for real-time analysis of photosynthesis at single-cell resolution.

Photoautotrophs' environmental responses have been extensively studied at the organism and ecosystem level. However, less is known about their photosynthesis at the single-cell level. This information is needed to understand photosynthetic acclimation processes, as light changes as it penetrates cells, layers of cells, or organs. Furthermore, cells within the same tissue may behave differently, being at different developmental/physiological stages. Here, we describe an approach for single-cell and subcellular photophysiology based on the customization of confocal microscopy to assess chlorophyll fluorescence quenching by the saturation pulse method. We exploit this setup to (1) reassess the specialization of photosynthetic activities in developing tissues of non-vascular plants; (2) identify a specific subpopulation of phytoplankton cells in marine photosymbiosis, which consolidate energetic connections with their hosts; and (3) examine the link between light penetration and photoprotection responses inside the different tissues that constitute a plant leaf anatomy.

The V-type ATPase enhances photosynthesis in marine phytoplankton and further links phagocytosis to symbiogenesis.

Diatoms, dinoflagellates, and coccolithophores are dominant groups of marine eukaryotic phytoplankton that are collectively responsible for the majority of primary production in the ocean.1 These phytoplankton contain additional intracellular membranes around their chloroplasts, which are derived from ancestral engulfment of red microalgae by unicellular heterotrophic eukaryotes that led to secondary and tertiary endosymbiosis.2 However, the selectable evolutionary advantage of these membranes and the physiological significance for extant phytoplankton remain poorly understood. Since intracellular digestive vacuoles are ubiquitously acidified by V-type H+-ATPase (VHA),3 proton pumps were proposed to acidify the microenvironment around secondary chloroplasts to promote the dehydration of dissolved inorganic carbon (DIC) into CO2, thus enhancing photosynthesis.4,5 We report that VHA is localized around the chloroplasts of centric diatoms and that VHA significantly contributes to their photosynthesis across a wide range of oceanic irradiances. Similar results in a pennate diatom, dinoflagellate, and coccolithophore, but not green or red microalgae, imply the co-option of phagocytic VHA activity into a carbon-concentrating mechanism (CCM) is common to secondary endosymbiotic phytoplankton. Furthermore, analogous mechanisms in extant photosymbiotic marine invertebrates6,7,8 provide functional evidence for an adaptive advantage throughout the transition from endosymbiosis to symbiogenesis. Based on the contribution of diatoms to ocean biogeochemical cycles, VHA-mediated enhancement of photosynthesis contributes at least 3.5 Gtons of fixed carbon per year (or 7% of primary production in the ocean), providing an example of a symbiosis-derived evolutionary innovation with global environmental implications.

Sexual Health-related Quality of Life in Women with Pulmonary Arterial Hypertension: Compensating for Loss.

Rationale: Health-related quality of life in patients with pulmonary arterial hypertension (PAH) has become increasingly important in disease management as numerous treatment options have improved prognosis and time to clinical worsening. Sexual health-related quality of life (SHRQoL) is poorly understood in patients with PAH, but previous work has shown that patients may face unrecognized challenges, especially related to parenteral prostanoid analogue therapies. Objectives: Using qualitative methods, to describe challenges and perspectives related to SHRQoL among women with PAH. Methods: We conducted 13 semistructured in-depth interviews at the Pulmonary Hypertension Association's International Pulmonary Hypertension Conference and Scientific Sessions among female attendees with World Symposium on Pulmonary Hypertension group 1 PAH. A coding structure using both deductive and inductive coding was developed to organize and analyze data using applied thematic analysis. Salient themes were identified and are presented here using summary and illustrative quotations. Results: Ninety-two percent (12 of 13) of participants reported declines in the frequency of sex after diagnosis of PAH. A significant portion (62% [8 of 13]) experienced fear of having sexual intercourse because of cardiopulmonary symptoms. All participants (100% [13 of 13]) reported compensatory behaviors/strategies during and around sexual intercourse; some participants on subcutaneous prostanoids also reported timing intercourse to coincide with infusion site changes and, as a result, interrupted treatment during this time. Participants reported changing positions during sex to reduce breathlessness, and some reported removing oxygen to avoid interrupting intimacy. Most participants endorsed negative body image related to their medications, external oxygen supplementation, and/or body weight fluctuations (54% [7 of 13]). Many participants revealed that they had never discussed sexual practices with healthcare professionals and desired increased communication and discussion with their providers. Conclusions: Women with PAH face significant burdens and challenges regarding SHRQoL. PAH therapies directly affect SHRQoL. Further targeted qualitative and quantitative studies are needed to better characterize and improve SHRQoL in patients with PAH.

HIV/AIDS-related knowledge, attitudes and perceptions: a cross-sectional household survey.

This study aimed to assess knowledge of and attitudes toward HIV/AIDS among a community in a semi-urban setting in Malaysia, to determine factors affecting perceptions toward people living with HIV in the community, and to provide baseline information for planning preventive measures against HIV/AIDS. This cross-sectional study was conducted in August 2009. Two hundred sixty-two household members were interviewed with a semi-structured questionnaire. Most respondents (232; 88.5%) had heard of HIV/AIDS. Only a few respondents (6; 2.6%) could correctly answer all the questionnaire items. Misconceptions about disease transmission were seen among surveyed participants, such as the belief HIV/AIDS can be contracted from saliva (104; 44.8%), mosquito bites (95; 40.9%) or casual touch (86; 37.1%). A multivariate linear regression model showed better perceptions towards people living with HIV depend on an improved knowledge of HIV/AIDS transmission. Current data emphasize the need to scale up HIV/AIDS education incorporating the mode of disease transmission.

A Rare Case of 4 Ps: Bilateral Pneumothoraces and Pneumomediastinum in Pneumocystis Pneumonia.

We report a case of Pneumocystis jirovecii pneumonia (PCP) complicated by bilateral pneumothoraces and pneumomediastinum in a non-human immunodeficiency virus (HIV)- infected patient. This unusual presentation exemplifies the differences in clinical course and presentation in non-HIV versus HIV-infected individuals, and the poor prognosis associated with PCP complicated by pneumothorax or pneumomediastinum. Providers should be aware of the high mortality in patients who develop one, and especially both complications.

Evolutionary links between intra- and extracellular acid-base regulation in fish and other aquatic animals.

The acid-base relevant molecules carbon dioxide (CO2 ), protons (H+ ), and bicarbonate (HCO3- ) are substrates and end products of some of the most essential physiological functions including aerobic and anaerobic respiration, ATP hydrolysis, photosynthesis, and calcification. The structure and function of many enzymes and other macromolecules are highly sensitive to changes in pH, and thus maintaining acid-base homeostasis in the face of metabolic and environmental disturbances is essential for proper cellular function. On the other hand, CO2 , H+ , and HCO3- have regulatory effects on various proteins and processes, both directly through allosteric modulation and indirectly through signal transduction pathways. Life in aquatic environments presents organisms with distinct acid-base challenges that are not found in terrestrial environments. These include a relatively high CO2 relative to O2 solubility that prevents internal CO2 /HCO3- accumulation to buffer pH, a lower O2 content that may favor anaerobic metabolism, and variable environmental CO2 , pH and O2 levels that require dynamic adjustments in acid-base homeostatic mechanisms. Additionally, some aquatic animals purposely create acidic or alkaline microenvironments that drive specialized physiological functions. For example, acidifying mechanisms can enhance O2 delivery by red blood cells, lead to ammonia trapping for excretion or buoyancy purposes, or lead to CO2 accumulation to promote photosynthesis by endosymbiotic algae. On the other hand, alkalinizing mechanisms can serve to promote calcium carbonate skeletal formation. This nonexhaustive review summarizes some of the distinct acid-base homeostatic mechanisms that have evolved in aquatic organisms to meet the particular challenges of this environment.

Expansion of the Major Facilitator Superfamily (MFS) to include novel transporters as well as transmembrane-acting enzymes.

The Major Facilitator Superfamily (MFS) is currently the largest characterized superfamily of transmembrane secondary transport proteins. Its diverse members are found in essentially all organisms in the biosphere and function by uniport, symport, and/or antiport mechanisms. In 1993 we first named and described the MFS which then consisted of 5 previously known families that had not been known to be related, and by 2012 we had identified a total of 74 families, classified phylogenetically within the MFS, all of which included only transport proteins. This superfamily has since expanded to 89 families, all included under TC# 2.A.1, and a few transporter families outside of TC# 2.A.1 were identified as members of the MFS. In this study, we assign nine previously unclassified protein families in the Transporter Classification Database (TCDB; http://www.tcdb.org) to the MFS based on multiple criteria and bioinformatic methodologies. In addition, we find integral membrane domains distantly related to partial or full-length MFS permeases in Lysyl tRNA Synthases (TC# 9.B.111), Lysylphosphatidyl Glycerol Synthases (TC# 4.H.1), and cytochrome b561 transmembrane electron carriers (TC# 5.B.2). Sequence alignments, overlap of hydropathy plots, compatibility of repeat units, similarity of complexity profiles of transmembrane segments, shared protein domains and 3D structural similarities between transport proteins were analyzed to assist in inferring homology. The MFS now includes 105 families.

Comparison of overexpansion capabilities and thrombogenicity at the side branch ostia after implantation of four different drug eluting stents.

Interventions in bifurcation lesions often requires aggressive overexpansion of stent diameter in the setting of long tapering vessel segment. Overhanging struts in front of the side branch (SB) ostium are thought to act as a focal point for thrombi formation and consequently possible stent thrombosis. This study aimed to evaluate the overexpansion capabilities and thrombogenicity at the SB ostia after implantation of four latest generation drug-eluting stents (DES) in an in-vitro bifurcation model. Four clinically available modern DES were utilized: one bifurcation dedicated DES (Bioss LIM C) and three conventional DES (Ultimaster, Xience Sierra, Biomime). All devices were implanted in bifurcation models with proximal optimization ensuring expansion before perfusing with porcine blood. Optical coherence tomography (OCT), immunofluorescence (IF) and scanning electron microscope analysis were done to determine thrombogenicity and polymer coating integrity at the over-expanded part of the stents. Computational fluid dynamics (CFD) was performed to study the flow disruption. OCT (p = 0.113) and IF analysis (p = 0.007) demonstrated lowest thrombus area at SB ostia in bifurcation dedicated DES with favorable biomechanical properties compared to conventional DES. The bifurcated DES also resulted in reduced area of high shear rate and maximum shear rate in the CFD analysis. This study demonstrated numerical differences in terms of mechanical properties and acute thrombogenicity at SB ostia between tested devices.

Structural and signaling requirements of the human melanocortin 4 receptor for MAP kinase activation.

In addition to its well known stimulation of cAMP production, the human melanocortin type 4 (hMC4) receptor recently has been shown to mediate p44/42 MAPK activation. This finding opens new questions about the structural and signaling mechanisms that connect the receptor to this alternate cell signaling pathway. Point mutants in the hMC4 receptor that have been associated with obesity were constructed and transfected into HEK 293 cells. Functional analyses then were done to determine if these mutations would similarly impact cAMP formation and p44/42 MAPK signaling. Whereas a D90N mutation in the second transmembrane domain and a D298A mutation in the seventh transmembrane domain impaired both cAMP formation and p44/42 MAPK activation, a more conservative D298N mutation retained cAMP formation but abolished p44/42 MAPK activation. The D298N mutation identified, for the first time, differential structural requirements of the hMC4 receptor for activation of the cAMP and p44/42 MAPK pathways. Furthermore, functional characterizations of a series of chimeric receptors combining the hMC4 receptor and the hMC3 subtype, a receptor that does not couple to p44/42 MAPK activation despite stimulating adenylyl cyclase, indicate that the hMC4 cytoplasmic tail is a necessary structural element for p44/42 MAPK signaling. Subsequent investigation of the signaling requirements for p44/42 MAPK activation demonstrated that the adenylyl cyclase inhibitor 2', 5'-dideoxyadenosine blocked agonist-induced p44/42 MAPK activation, but the PKA inhibitor Rp cAMPS did not. Taken together, these data indicate that cAMP is required, but not sufficient for p44/42 MAPK activation and suggest structural elements required for hMC4 receptor signaling.

Identification of structural determinants for G protein-independent activation of mitogen-activated protein kinases in the seventh transmembrane domain of the angiotensin II type 1 receptor.

Although the intrareceptor mechanisms whereby the angiotensin II (AngII) type 1 receptor activates phospholipase C (PLC) have been extensively investigated, analogous studies of signaling through mitogen-activated protein kinases (MAPK) have been lacking. We investigated MAPK activation and traditional G(q)/PLC signaling in transfected cells using AngII and the signaling selective agonist [Sar(1),Ile(4),Ile(8)] AngII (SII). SII stimulated MAPK without inositol trisphosphate (IP(3)) production and thereby stabilizes an activated receptor state linked to G protein-independent MAPK signaling. Using receptor mutagenesis, we focused on the seventh transmembrane domain and identified three key residues-Tyr(292), Phe(293), and Thr(287). At least three distinct activated states were revealed: 1) an AngII-stabilized state linked to G(q)/PLC signaling, 2) an AngII-stabilized state connected to G protein-independent MAPK activation, and 3) a SII-stabilized state associated with G protein-independent MAPK signaling. The mutant Y292F failed to exhibit AngII-induced IP(3) turnover yet remained capable of AngII-induced MAPK activation. SII failed to stimulate MAPK in Y292F-transfected cells. Thus, Tyr(292) is a key epitope for activated states 1 and 3 but not required for activated state 2. Although the F293L mutant retained normal AngII responses, it also showed an IP(3) response to SII, indicating that Phe(293) may be involved in constraining the receptor to its inactive state. Mutations of Thr(287) abolished all SII-induced signaling without affecting any AngII responses. Thr(287) therefore represents a key residue for a SII-stabilized activated state. Taken together, the data identified a novel structural requirement (Thr(287)) for the SII-stabilized activated state and redefined the mechanistic roles for Tyr(292) and Phe(293).