Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin

Transitory binding between photoactivated rhodopsin (Rho* or Meta II) and the G protein transducin (Gt-GDP) is the first step in the visual signaling cascade. Light causes photoisomerization of the 11-cis-retinylidene chromophore in rhodopsin (Rho) to all-trans-retinylidene, which induces conformational changes that allow Gt-GDP to dock onto the Rho* surface. GDP then dissociates from Gt, leaving a transient nucleotide-empty Rho*-Gt(e) complex before GTP becomes bound, and Gt-GTP then dissociates from Rho*. Further biochemical advances are required before structural studies of the various Rho*-Gt complexes can be initiated. Here, we describe the isolation of n-dodecyl-beta-maltoside solubilized, stable, functionally active, Rho*-Gt(e), Rho(e)*-Gt(e), and 9-cis-retinal/11-cis-retinal regenerated Rho-Gt(e) complexes by sucrose gradient centrifugation. In these complexes, Rho* spectrally remained in its Meta II state, and Gt(e) retained its ability to interact with GTPgammaS. Removal of all-trans-retinylidene from Rho*-Gt(e) had no effect on the stability of the Rho(e)*-Gt(e) complex. Moreover, opsin in the Rho(e)*-Gt(e) complex with an empty nucleotide-binding pocket in Gt and an empty retinoid-binding pocket in Rho was regenerated up to 75% without complex dissociation. These results indicate that once Rho* couples with Gt, the chromophore plays a minor role in stabilizing this complex. Moreover, in complexes regenerated with 9-cis-retinal/11-cis-retinal, Rho retains a conformation similar to Rho* that is stabilized by Gt(e) apo-protein.

The chemokine CCL20 and its receptor CCR6 in human malignancy with focus on colorectal cancer

Chemokines are a superfamily of small chemotactic cytokines, which interact with their G-protein-coupled receptors. These interactions regulate multiple physiological functions, particularly tissue architecture and compartment-specific migration of white blood cells. It has been found that the chemokine/chemokine receptor system has been utilized by cancer cells for migration and metastasis. The chemokine receptor CCR6 is expressed in colorectal cancer and several other cancer types, and stimulation by its physiological chemokine ligand CCL20 has been reported to promote cancer cell proliferation and migration in vitro. Moreover, CCR6/CCL20 interactions apparently play a role in organ selective liver metastasis of colorectal cancer. Here, we review the literature on expression patterns of CCL20 and CCR6 and their physiological interactions as well as the currently presumed role of CCR6 and CCL20 in the formation of colorectal cancer liver metastasis, providing a potential basis for novel treatment strategies.

Anticancer activity of anandamide in human cutaneous melanoma cells

Cannabinoids are implicated in the control of cell proliferation, but little is known about the role of the endocannabinoid system in human malignant melanoma. This study was aimed at characterizing the in vitro antitumor activity of anandamide (AEA) in A375 melanoma cells. The mRNA expression of genes that code for proteins involved in the metabolism and in the mechanism of AEA action was assessed by RT-PCR. Cell viability was tested using WST-1 assay and the apoptotic cell death was determined by measuring caspase 3/7 activities. A375 cells express high levels of fatty acid amide hydrolase (FAAH), cyclooxygenase (COX)-2, cannabinoid receptor 1 (CB1), transient receptor potential cation channel subfamily V member 1 (TRPV1) and G-protein-coupled receptor 55 (GPR55) genes. AEA induced a concentration-dependent cytotoxicity with an IC50 of 5.8±0.7 µM and such an effect was associated to a caspase-dependent apoptotic pathway. AEA cytotoxicity was potentiated by FAAH inhibition (2-fold increase, p<0.05) and mitigated by COX-2 or lipoxygenase (LOX) inhibition (5- and 3-fold decrease, respectively; p<0.01). Blocking CB1 receptors partially decreased AEA cytotoxicity, whereas selective antagonism on the TRPV1 barely affected the mechanism of AEA action. Finally, methyl-β-cyclodextrin, a membrane cholesterol depletory, completely reversed the cytotoxicity induced by the selective GPR55 agonist, O-1602, and AEA. Overall, these findings demonstrate that AEA induces cytotoxicity against human melanoma cells in the micromolar range of concentrations through a complex mechanism, which involves COX-2 and LOX-derived product synthesis and CB1 activation. Lipid raft modulation, probably linked to GPR55 activation, might also have a role.

Clinical, genetic, and functional characterization of adrenocorticotropin receptor mutations using a novel receptor assay.

The ACTH receptor (MC2R) is expressed predominantly in the adrenal cortex, but is one of five G protein-coupled, seven-transmembrane melanocortin receptors (MCRs), all of which bind ACTH to some degree. Testing of MC2R activity is difficult because most cells express endogenous MCRs; hence, ACTH will elicit background activation of assayable reporter systems. Inactivating mutations of MC2R lead to hereditary unresponsiveness to ACTH, also known as familial glucocorticoid deficiency (FGD). These patients are usually seen in early childhood with very low cortisol concentrations, normal mineralocorticoids, hyperpigmentation, and increased bodily growth. Several MC2R mutations have been reported in FGD, but assays of the activities of these mutants are cumbersome. We saw two patients with typical clinical findings of FGD. Genetic analysis showed that patient 1 was homozygous for the mutation R137W, and patient 2 was a compound heterozygote for S74I and Y254C. We tested the activity of these mutations in OS-3 cells, which are unresponsive to ACTH but have intact downstream cAMP signal transduction. OS-3 cells transfected with a cAMP-responsive luciferase reporter plasmid (pCREluc) were unresponsive to ACTH, but cotransfection with a vector expressing human MC2R increased luciferase activity more than 40-fold. Addition of ACTH to cells cotransfected with the pCREluc reporter and wild-type MC2R activated luciferase expression with a 50% effective concentration of 5.5 x 10(-9) M ACTH, which is similar to previously reported values. By contrast, the MC2R mutant R137W had low activity, and the S74I or Y254C mutants elicited no measurable response. This assay provides excellent sensitivity in an easily assayed transient transfection system, providing a more rapid and efficient measurement of ACTH receptor activity.

THE ROLE OF A2B ADENOSINE RECEPTOR SIGNALING IN ADENOSINE DEPENDENT LUNG DISEASE

Chronic lung diseases and acute lung injuries are two distinctive pulmonary disorders that result in significant morbidity and mortality. Adenosine is a signaling nucleoside generated in response to injury and can serve both protective and destructive functions in tissues and cells through interaction with four G-protein coupled adenosine receptors: A1R, A2AR, A2BR, and A3R. However, the relationship between these factors is poorly understood. Recent findings suggest the A2BR has been implicated in the regulation of both chronic lung disease and acute lung injury. The work presented in this dissertation utilized the adenosine deaminase-deficient mouse model and the bleomycin-induced pulmonary injury model to determine the distinctive roles of the A2BR at different stages of the disease. Results demonstrate that the A2BR plays a protective role in attenuating vascular leakage in acute lung injuries and a detrimental role at chronic stages of the disease. In addition, tissues from patients with chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis were utilized to examine adenosine metabolism and signaling in chronic lung diseases. Results demonstrate that components of adenosine metabolism and signaling are altered in a manner that promotes adenosine production and signaling in the lungs of these patients. Furthermore, this study provides the first evidence that A2BR signaling can promote the production of inflammatory and fibrotic mediators in patients with these disorders. Taken together, these findings suggest that the A2BR may have a bi-phasic effect at different stages of lung disease. It is protective in acute injury, whereas pro-inflammatory and pro-fibrotic at the chronic stage. Patients with acute lung injury or chronic lung disease may both benefit from adenosine and A2BR-based therapeutics.

THE EXPRESSION AND CELLULAR LOCALIZATION OF CC-CHEMOKINE RECEPTOR 5 (CCR5) AFTER TRAUMATIC BRAIN INJURY

Traumatic brain injury results from a primary insult and secondary events that together result in tissue injury. This primary injury occurs at the moment of impact and damage can include scalp laceration, skull fraction, cerebral contusions and lacerations as well as intracranial hemorrhage. Following the initial insult, a delayed response occurs and is characterized by hypoxia, ischemia, cerebral edema, and infection. During secondary brain injury, a series of neuroinflammatory events are triggered that can produce additional damage but may also help to protect nervous tissue from invading pathogens and help to repair the damaged tissue. Brain microglia and astrocytes become activated and migrate to the site of injury where these cells secrete immune mediators such as cytokines and chemokines. CC-chemokine receptor 5 (CCR5) is a member of the CC chemokine receptor family of seven transmembrane G protein coupled receptors. CCR5 is expressed in the immune system and is found in monocytes, leukoctyes, memory T cells, and immature dendritic cells. Upon binding to its ligands, CCR5 functions in the chemotaxis of these immune cells to the site of inflammation. In the CNS, CCR5 and its ligands are expressed in multiple cell types. In this study, I investigated whether CCR5 expression is altered in brain after traumatic brain injury. I examined the time course of CCR5 protein expression in cortex and hippocampus using quantitative western analysis of tissues from injured rat brain after mild impact injury. In addition, I also investigated the cellular localization of CCR5 before and after brain injury using confocal microscopy. I have observed that after brain injury CCR5 is upregulated in a time dependent manner in neurons of the parietal cortex and hippocampus. The absence of CCR5 expression in microglia and its delayed expression in neurons after injury suggests a role for CCR5 in neuronal survival after injury.

THE EXPRESSION AND CELLULAR LOCALIZATION OF CC-CHEMOKINE RECEPTOR 5 (CCR5) AFTER TRAUMATIC BRAIN INJURY

Traumatic brain injury results from a primary insult and secondary events that together result in tissue injury. This primary injury occurs at the moment of impact and damage can include scalp laceration, skull fraction, cerebral contusions and lacerations as well as intracranial hemorrhage. Following the initial insult, a delayed response occurs and is characterized by hypoxia, ischemia, cerebral edema, and infection. During secondary brain injury, a series of neuroinflammatory events are triggered that can produce additional damage but may also help to protect nervous tissue from invading pathogens and help to repair the damaged tissue. Brain microglia and astrocytes become activated and migrate to the site of injury where these cells secrete immune mediators such as cytokines and chemokines. CC-chemokine receptor 5 (CCR5) is a member of the CC chemokine receptor family of seven transmembrane G protein coupled receptors. CCR5 is expressed in the immune system and is found in monocytes, leukoctyes, memory T cells, and immature dendritic cells. Upon binding to its ligands, CCR5 functions in the chemotaxis of these immune cells to the site of inflammation. In the CNS, CCR5 and its ligands are expressed in multiple cell types. In this study, I investigated whether CCR5 expression is altered in brain after traumatic brain injury. I examined the time course of CCR5 protein expression in cortex and hippocampus using quantitative western analysis of tissues from injured rat brain after mild impact injury. In addition, I also investigated the cellular localization of CCR5 before and after brain injury using confocal microscopy. I have observed that after brain injury CCR5 is upregulated in a time dependent manner in neurons of the parietal cortex and hippocampus. The absence of CCR5 expression in microglia and its delayed expression in neurons after injury suggests a role for CCR5 in neuronal survival after injury.

THE ROLE OF TYROSINE PHOSPHORYLATION IN THE FUNCTIONS OF THE TUMOR SUPPRESSOR GPRC5A

The retinoic acid inducible G protein coupled receptor family C group 5 type A (GPRC5A) is expressed preferentially in normal lung tissue but its expression is suppressed in the majority of human non-small cell lung cancer cell lines and tissues. This differential expression has led to the idea that GPRC5A is a potential tumor suppressor. This notion was supported by the finding that mice with a deletion of the Gprc5a gene develop spontaneous lung tumors. However, there are various tumor cell lines and tissue samples, including lung, that exhibit higher GPRC5A expression than normal tissues and some reports by other groups that GPRC5A transfection increased cell growth and colony formation. Obviously, GPRC5A has failed to suppress the development of the tumors and the growth of the cell lines where its expression is not suppressed. Since no mutations were detected in the coding sequence of GPRC5A in 20 NSCLC cell lines, it’s possible that GPRC5A acts as a tumor suppressor in the context of some cells but not in others. Alternatively, we raised the hypothesis that the GPRC5A protein may be inactivated by posttranslational modification(s) such as phosphorylation. It is well established that Serine/Threonine phosphorylation of G protein coupled receptors leads to their desensitization and in a few cases Tyrosine phosphorylation of GPCRs has been linked to internalization. Others reported that GPRC5A can undergo tyrosine phosphorylation in the cytoplasmic domain after treatment of normal human mammary epithelial cells (HMECs) with epidermal growth factor (EGF) or Heregulin. This suggested that GPRC5A is a substrate of EGFR. Therefore, we hypothesized that tyrosine phosphorylation of GPRC5A by activation of EGFR signaling may lead to its inactivation. To test this hypothesis, we transfected human embryo kidney (HEK) 293 cells with GPRC5A and EGFR expression vectors and confirmed that GPRC5A can be tyrosine phosphorylated after activation of EGFR by EGF. Further, we found that EGFR and GPRC5A can interact either directly or through other proteins and that inhibition of the EGFR kinase activity decreased the phosphorylation of GPRA5A and the interaction between GPRC5A and EGFR. In c-terminal of GPRC5A, There are four tyrosine residues Y317, Y320, Y347, Y350. We prepared GPRC5A mutants in which all four tyrosine residues had been replaced by phenylalanine (mutant 4F) or each individual Tyr residue was replaced by Phe and found that Y317 is the major site for EGFR mediated phosphorylation in the HEK293T cell line. We also found that EGF can induce GPRC5A internalization both in H1792 transient and stable cell lines. EGF also partially inactivates the suppressive function of GPRC5A on cell invasion activity and anchorage-independent growth ability of H1792 stable cell lines. These finding support our hypothesis that GPRC5A may be inactivated by posttranslational modification- tyrosine phosphorylation.

Naive B-cell trafficking is shaped by local chemokine availability and LFA-1-independent stromal interactions.

It is not known how naive B cells compute divergent chemoattractant signals of the T-cell area and B-cell follicles during in vivo migration. Here, we used two-photon microscopy of peripheral lymph nodes (PLNs) to analyze the prototype G-protein-coupled receptors (GPCRs) CXCR4, CXCR5, and CCR7 during B-cell migration, as well as the integrin LFA-1 for stromal guidance. CXCR4 and CCR7 did not influence parenchymal B-cell motility and distribution, despite their role during B-cell arrest in venules. In contrast, CXCR5 played a nonredundant role in B-cell motility in follicles and in the T-cell area. B-cell migration in the T-cell area followed a random guided walk model, arguing against directed migration in vivo. LFA-1, but not α4 integrins, contributed to B-cell motility in PLNs. However, stromal network guidance was LFA-1 independent, uncoupling integrin-dependent migration from stromal attachment. Finally, we observed that despite a 20-fold reduction of chemokine expression in virus-challenged PLNs, CXCR5 remained essential for B-cell screening of antigen-presenting cells. Our data provide an overview of the contribution of prototype GPCRs and integrins during naive B-cell migration and shed light on the local chemokine availability that these cells compute.

Regulation of the gastrin-releasing peptide/bombesin receptor

Gastrin-releasing peptide (GRP) and other bombesin-like peptides stimulate hormone secretion and cell proliferation by binding to specific G-protein-coupled receptors. Three studies were performed to identify potential mechanisms involved in GRP/bombesin receptor regulation.^ Although bombesin receptors are localized throughout the gastrointestinal tract, few gastrointestinal cell lines are available to study bombesin action. In the first study, the binding and function of bombesin receptors in the human HuTu-80 duodenal cancer cell line were characterized. ($\sp{125}$I-Tyr$\sp4$) bombesin bound with high affinity to a GRP-preferring receptor. Bombesin treatment increased IP$\sb3$ production, but had no effect on cell proliferation. Similar processing of ($\sp{125}$I-Tyr$\sp4$) bombesin and of GRP-receptors was observed in HuTu-80 cells and Swiss 3T3 fibroblasts, a cell line which mitogenically responds to bombesin. Therefore, the lack of a bombesin mitogenic effect in HuTu-80 cells is not due to unusual processing of ($\sp{125}$I-Tyr$\sp4$) bombesin or rapid GRP-receptor down-regulation.^ In the second study, a bombesin antagonist was developed to study the processing and regulatory events after antagonist binding. As previously shown, receptor bound agonist, ($\sp{125}$I-Tyr$\sp4$) bombesin, was rapidly internalized and degraded in chloroquine-sensitive compartments. Interestingly, receptor-bound antagonist, ($\sp{125}$I-D-Tyr$\sp6$) bombesin(6-13)PA was not internalized, but degraded at the cell-surface. In contrast to bombesin, (D-Tyr$\sp6$) bombesin(6-13)PA treatment did not cause receptor internalization. Together these results demonstrate that receptor regulation and receptor-mediated processing of antagonist is different from that of agonist.^ Bombesin receptors undergo acute desensitization. By analogy to other G-protein-coupled receptors, a potential desensitization mechanism may involve receptor phosphorylation. In the final study, $\sp{32}$P-labelled Swiss 3T3 fibroblasts and CHO-mBR1 cells were treated with bombesin and the GRP-receptor was immunoprecipitated. In both cell lines, bombesin treatment markedly stimulated GRP-receptor phosphorylation. Furthermore, bombesin-stimulated GRP-receptor phosphorylation occurred within the same time period as bombesin-stimulated desensitization, demonstrating that these two processes are correlated.^ In conclusion, these studies of GRP-receptor regulation further our understanding of bombesin action and provide insight into G-protein-coupled receptor regulation in general. ^

Study of retinoic acid signaling in cancer cells: Cloning and characterization of retinoic acid responsive genes

Retinoids such as all-trans-retinoic acid (ATRA) are promising agents for cancer chemoprevention and therapy. ATRA can cause growth inhibition, induction of differentiation and apoptosis of a variety of cancer cells. These effects are thought to be mediated by nuclear retinoids receptors which are involved in ligand-dependent transcriptional activation of downstream target genes. Using differential display, we identified several retinoic acid responsive genes in the head and neck squamous carcinoma cells and lung cancer cells, including tissue type transglutaminase, cytochrome P450-related retinoic acid hydroxylase, and a novel gene, designated RAIG1. RAIG1 has two transcripts of 2.4 and 6.8 kbp, respectively, that are generated by alternative selection of polyadenylation sites. Both transcripts have the same open reading frame that encodes a protein comprised of 357 amino acid residues. The deduced RAIG1 protein sequence contains seven transmembrane domains, a signature structure of G protein-coupled receptors. RAIG1 mRNA is expressed at high level in fetal and adult lung tissues. Induction of RAIG1 expression by ATRA is rapid and dose-dependent. A fusion protein of RAIG1 and the green fluorescent protein was localized in the cell surface membrane and perinuclear vesicles in transiently transfected cells. The locus for RAIG1 gene was mapped to a region between D12S358 and D12S847 on chromosome 12p12.3-p13. Our study of the novel retinoic acid induced gene RAIG1 provide evidence for a possible interaction between retinoid and G protein signaling pathways.^ We further examined RAIG1 expression pattern in a panel of 84 cancer cell lines of different origin. The expression level varies greatly from very high to non-detectable. We selected a panel of different cancer cells to study the effects of retinoids and other differentiation agents. We observed: (1) In most cases, retinoids (including all-trans retinoic acid, 4HPR, CD437) could induce the expression of RAIG-1 in cells from cancers of the breast, colon, head and neck, lung, ovarian and prostate. (2) Compare to retinoids, butyrate is often a more potent inducer of RAIG-1 expression in many cancer cells. (3) Butyrate, Phenylacetate butyrate, (R)P-Butyrate and (S)P-Butyrate have different impact on RAIG1 expression which varies among different cell lines. Our results indicate that retinoids could restore RAIG1 expression that is down-regulated in many cancer cells.^ A mouse homologous gene, mRAIG1, was cloned by 5$\sp\prime$ RACE reaction. mRAIG1 cDNA has 2105 bp and shares 63% identity with RAIG1 cDNA. mRAIG1 encodes a polypeptide of 356 amino acid which is 76% identity with RAIG1 protein. mRAIG1 protein also has seven transmembrane domains which are structurally identical to those of RAIG1 protein. Only one 2.2 kbp mRAIG1 transcript could be detected. The mRAIG1 mRNA is also highly expressed in lung tissue. The expression of mRAIG1 gene could be induced by ATRA in several mouse embryonal carcinoma cells. The induction of mRAIG1 expression is associated with retinoic acid-induced neuroectoderm differentiation of P19 cells. Similarity in cDNA and protein sequence, secondary structure, tissue distribution and inducible expression by retinoic acid strongly suggest that the mouse gene is the homologue of the human RAIG1 gene. ^

Memo has a novel role in S1P signaling and crucial for vascular development

Memo is a conserved protein that was identified as an essential mediator of tumor cell motility induced by receptor tyrosine kinase activation. Here we show that Memo null mouse embryonic fibroblasts (MEFs) are impaired in PDGF-induced migration and this is due to a defect in sphingosine-1-phosphate (S1P) signaling. S1P is a bioactive phospholipid produced in response to multiple stimuli, which regulates many cellular processes. S1P is secreted to the extracellular milieu where it exerts its function by binding a family of G-protein coupled receptors (S1PRs), causing their activation in an autocrine or paracrine manner. The process, termed cell-autonomous S1PR signaling, plays a role in survival and migration. Indeed, PDGF uses cell-autonomous S1PR signaling to promote cell migration; we show here that this S1P pathway requires Memo. Using vascular endothelial cells (HUVECs) with Memo knock-down we show that their survival in conditions of serum-starvation is impaired. Furthermore, Memo loss in HUVECs causes a reduction of junctional VE-cadherin and an increase in sprout formation. Each of these phenotypes is rescued by S1P or S1P agonist addition, showing that Memo also plays an important role in cell-autonomous S1PR signaling in endothelial cells. We also produced conventional and endothelial cell-specific conditional Memo knock-out mouse strains and show that Memo is essential for embryonic development. Starting at E13.5 embryos of both strains display bleeding and other vascular problems, some of the phenotypes that have been described in mouse strains lacking S1PRs. The essential role of Memo in embryonic vascular development may be due in part to alterations in S1P signaling. Taken together our results show that Memo has a novel role in the S1P pathway and that Memo is needed to promote cell-autonomous S1PR activation.

Regulation of androgen biosynthesis - A short review and preliminary results from the hyperandrogenic starvation NCI-H295R cell model

Regulation of androgen production is poorly understood. Adrenarche is the physiologic event in mid-childhood when the adrenal zona reticularis starts to produce androgens through specific expression of genes for enzymes and cofactors necessary for androgen synthesis. Similarly, expression and activities of same genes and products are deregulated in hyperandrogenic disorders such as the polycystic ovary syndrome (PCOS). Numerous studies revealed involvement of several signaling pathways stimulated through G-protein coupled receptors or growth factors transmitting their effects through cAMP- or non-cAMP-dependent signaling. Overall a complex network regulates androgen synthesis targeting involved genes and proteins at the transcriptional and post-translational levels. Newest players in the field are the DENND1A gene identified in PCOS patients and the MAPK14 which is the kinase phosphorylating CYP17 for enhanced lyase activity. Next generation sequencing studies of PCOS patients and transcriptome analysis of androgen producing tissues or cell models provide newer tools to identify modulators of androgen synthesis.

The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

Molecular mechanisms of prostacyclin receptor signaling: The intracellular domains in G protein coupling

To better understand the mechanisms of how the human prostacyclin receptor (1P) mediates vasodilation and platelet anti-aggregation through Gs protein coupling, a strategy integrating multiple approaches including high resolution NMR experiments, synthetic peptide, fluorescence spectroscopy, molecular modeling, and recombinant protein was developed and used to characterize the structure/function relationship of important segments and residues of the IP receptor and the α-subunit of the Gs protein (Gαs). The first (iLP1) and third (iLP3) intracellular loops of the IP receptor, as well as the Gαs C-terminal domain, relevant to the Gs-mediated IP receptor signaling, were first identified by observation of the effects of the mini gene-expressed corresponding protein segments in HEK293 cells which co-expressed the receptor and Gαs. Evidence of the IP iLP1 domain interacted with the Gαs C-terminal domain was observed by fluorescence and NMR spectroscopic studies using a constrained synthetic peptide, which mimicked the IP iLP1 domain, and the synthetic peptide, which mimicked Gαs C-terminal domain. The solution structural models and the peptide-peptide interaction of the two synthetic protein segments were determined by high resolution NMR spectroscopy. The important residues in the corresponding domains of the IP receptor and the Gαs predicted by NMR chemical shift mapping were used to guide the identification of their protein-protein interaction in cells. A profile of the residues Arg42 - Ala48 of the IP iLP1 domain and the three residues Glu392 ∼ Leu394 of the Gαs C-terminal domain involved in the IP/Gs protein coupling were confirmed by recombinant proteins. The data revealed an intriguing speculation on the mechanisms of how the signal of the ligand-activated IP receptor is transmitted to the Gs protein in regulating vascular functions and homeostasis, and also provided substantial insights into other prostanoid receptor signaling. ^