Ligand-stimulated internalization of a Dictyostelium discoideum G protein-coupled cAMP receptor

The social amoeba, Dictyostelium discoideum, undergoes a remarkable starvation-induced program of development that transforms a population of unicellular amoebae into a fruiting body composed of resistant spores suspended on a stalk. During this development, secreted cAMP drives chemotaxis of the amoebae, leading to their aggregation, and subsequent differentiation and morphogenesis. Four sequentially expressed G protein-coupled receptors (GPCRs) for cAMP play critical roles in this process. The first of these, cAR1, is essential for aggregation as it mediates chemotaxis as well as the propagation of secreted cAMP waves throughout aggregating populations. Ligand-induced internalization has been shown to regulate a variety of GPCRs. However, little was known at the outset of this study about the role of internalization in the regulation of cAR1 function or, for that matter, in developmental systems in general. For this study, cAMP-induced cAR1 internalization was assessed by measuring (1) the reduction of cell surface binding sites for [ 3H]cAMP and (2) the redistribution of YFP-tagged receptors to the cell's interior, cAMP was found to induce little or no loss of ligand binding (LLB) in vegetative cells. However, the ability to induce LLB increased progressively over the initial 6 hrs of development, reaching ∼70% in cells undergoing aggregation. Despite these reductions in surface binding, detectable cAR1-YFP redistribution could be induced by cAMP only after the cells reached the mound stage (10 hrs) and was found to occur naturally by the ensuing slug stage (18 hrs). Site-directed substitution of a cluster of 5 serines in the receptor's cytoplasmic tail that was previously shown to be the principal site of cAMP-induced cAR1 phosphorylation impaired both LLB and receptor redistribution and furthermore resulted in mound-stage developmental arrest, suggesting that phosphorylation of cAR1 is a prerequisite for its internalization and that cAR1 internalization is required for post-aggregative development. To assess the involvement of clathrin mediated endocytosis, Dictyostelium cells lacking the clathrin light chain gene (clc-) or either of two dynamin genes were examined and found to be defective in LLB and, in the case of clc- cells, also cAR1 redistribution and turnover. Furthermore, cAR1 overexpression in clc- cells (like the serine mutant in wild-type cells) promoted developmental arrest in mounds. The mound-arrest phenotype was also recapitulated in a wild-type background by the specific expression of cAR1 in prestalk cells (but not prespore cells), suggesting that development depends critically on internalization and clearance of cAR1 from these cells. Persistent cAR1 expression following aggregation was found to be associated with aberrant expression of prestalk and prespore genes, which may adversely affect development in the prestalk cell lineage. The PI3 kinase-TORC2 signal transduction pathway, known to be important for Dictyostelium chemotaxis and internalization of yeast pheromone receptors, was examined using chemical inhibitors and null cells and found to be necessary for cAR1 internalization. In conclusion, cAR1 was shown to be similar to other GPCRs in that its internalization depends on phosphorylation of cytoplasmic domain serines, utilizes clathrin and dynamin, and involves the TORC2 complex. In addition, the findings presented here that cAR1 internalization is both developmentally regulated and required for normal development represent a novel regulatory paradigm that might pertain to other GPCRs known to play important roles in the development of humans and other metazoans. ^

Specificity of somatostatin receptor and G -protein interactions as characterized by somatostatin receptor subtype specific antibodies

The neuropeptide somatostatin is a widely distributed general inhibitor of endocrine, exocrine, gastrointestinal and neural functions. The biological actions of somatostatin are initiated by interaction with high affinity, plasma membrane somatostatin receptors (sst receptors). Five sst receptor subtypes have been cloned and sequence analysis shows they are all members of the G protein coupled receptor superfamily. The G proteins play a pivotal role in sst receptor signal transduction and the specificity of somatostatin receptor-G protein coupling defines the possible range of cellular responses. However, the data for endogenous sst receptor and G protein coupling is very limited, and even when it is available, the sst receptor subtypes involved in G protein coupling and signal transduction are unknown due to the expression of multiple sst receptor subtypes in target cell lines or tissues of somatostatin.^ In an effort to characterize each individual sst receptor subtypes, antisera against unique C-terminal regions of different sst receptor subtypes have been developed in our lab. In this report, antisera made against the sst1, sst2A and sst4 receptors are characterized. They are highly specific to their corresponding receptors and efficiently immunoprecipitate the sst receptors. Using these antibodies, the cell lines expressing these sst receptor subtypes were identified with both immunoprecipitation and Western blot methods. The development of sst receptor subtype specific antibodies make it possible to determine the specificity of the sst receptor subtype and G protein coupling in target cells or tissues expressing multiple sst receptors, two questions were addressed by this thesis: (1) whether different cellular environments affect receptor subtype and G protein coupling; (2) whether different sst receptors couple to different G proteins in similar cellular environments.^ Taken together our findings, both sst1 and sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells, G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in GH$\sb4$C$\sb1$ cells. Further, sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in AR4-2J cells while sst4 receptors couple with G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells. Therefore, the G protein coupling of the same sst receptors in different cell lines is basically similar in that they all couple with multiple $\alpha$-subunits of the G$\rm \sb{i}$ proteins, suggesting cellular environment has little effect on receptor and G protein coupling. Moreover, different sst receptors have similar G protein coupling specificities in the same cell line, suggesting components other than receptor and G$\alpha$ subunits in the signal transduction pathways may contribute to specific functions of each sst receptor subtype. This series of experiments represent a novel approach in dissecting signal transduction pathways and may have general application in the field. Furthermore, this is the first systematic study of sst receptor subtype and G protein $\alpha$-subunit interaction in both transfected cells and in normal cell lines. The information generated will be very useful in our understanding of sst receptor signal transduction pathways and in directing future sst receptor research. ^

MODELLING β2AR REGULATION

The β2 adrenergic receptor (β2AR) regulates smooth muscle relaxation in the vasculature and airways. Long- and Short-acting β-agonists (LABAs/SABAs) are widely used in treatment of chronic obstructive pulmonary disorder (COPD) and asthma. Despite their widespread clinical use we do not understand well the dominant β2AR regulatory pathways that are stimulated during therapy and bring about tachyphylaxis, which is the loss of drug effects. Thus, an understanding of how the β2AR responds to various β-agonists is crucial to their rational use. Towards that end we have developed deterministic models that explore the mechanism of drug- induced β2AR regulation. These mathematical models can be classified into three classes; (i) Six quantitative models of SABA-induced G protein coupled receptor kinase (GRK)-mediated β2AR regulation; (ii) Three phenomenological models of salmeterol (a LABA)-induced GRK-mediated β2AR regulation; and (iii) One semi-quantitative, unified model of SABA-induced GRK-, protein kinase A (PKA)-, and phosphodiesterase (PDE)-mediated regulation of β2AR signalling. The various models were constrained with all or some of the following experimental data; (i) GRK-mediated β2AR phosphorylation in response to various LABAs/SABAs; (ii) dephosphorylation of the GRK site on the β2AR; (iii) β2AR internalisation; (iv) β2AR recycling; (v) β2AR desensitisation; (vi) β2AR resensitisation; (vii) PKA-mediated β2AR phosphorylation in response to a SABA; and (viii) LABA/SABA induced cAMP profile ± PDE inhibitors. The models of GRK-mediated β2AR regulation show that plasma membrane dephosphorylation and recycling of the phosphorylated β2AR are required to reconcile with the measured dephosphorylation kinetics. We further used a consensus model to predict the consequences of rapid pulsatile agonist stimulation and found that although resensitisation was rapid, the β2AR system retained the memory of prior stimuli and desensitised much more rapidly and strongly in response to subsequent stimuli. This could explain tachyphylaxis of SABAs over repeated use in rescue therapy of asthma patients. The LABA models show that the long action of salmeterol can be explained due to decreased stability of the arrestin/β2AR/salmeterol complex. This could explain long action of β-agonists used in maintenance therapy of asthma patients. Our consensus model of PKA/PDE/GRK-mediated β2AR regulation is being used to identify the dominant β2AR desensitisation pathways under different therapeutic regimens in human airway cells. In summary our models represent a significant advance towards understanding agonist-specific β2AR regulation that will aid in a more rational use of the β2AR agonists in the treatment of asthma.

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.

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. ^

Population and molecular evolution studies on the Melanocortin 1 receptor locus implicate its role in human pigmentation variation

Human pigmentation is a complex trait with the observed variation caused by the varied production of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) by the melanocytes. The melanocortin 1 receptor (MC1R), a G protein-coupled receptor expressed in the melanocytes, is a regulator eu- and phaeomelanin synthesis, and MC1R mutations causing skin and coat color changes are known in many mammals. To understand the role of MC1R in human pigmentation variation, I have sequenced the MC1R gene in 121 individuals sampled from world populations. In addition, I have sequenced the MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon to study the evolution of MC1R and to infer the ancestral human MC1R sequence. The ancestral MC1R sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. To further evaluate the role of MC1R variants in human pigmentation variation, I have combined these molecular evolution and population studies with functional assays on MC1R variants and primate MC1Rs. ^

LOSS OF GPRC5A ENHANCES SURVIVAL IN NORMAL AND MALIGNANT LUNG EPITHELIAL CELLS BY ELICITING PERSISTENT STAT3 ACTIVATION INDUCED BY AUTOCRINE LIF

Signal transduction and activator of transcription 3 (Stat3) is activated by cytokines and growth factors in many cancers. Persistent activation of Stat3 plays important role in cell growth, survival, and transformation through regulating its targeted genes. Previously, we found that mice with a deletion of the G protein-coupled receptor, family C, group 5, member a (Gprc5a) gene develop lung tumors indicating that Gprc5a is a tumor suppressor. In the present study, we examined he mechanism of Gprc5a-mediated tumor suppression. We found that epithelial cells from Gprc5a knockout mouse lung (Gprc5a-/- cells) survive better in vitro in medium deprived of exogenous growth factors and form more colonies in semi-solid medium than their counterparts from wildtype mice (Gprc5a+/+ cells). The phosphorylation of tyrosine 705 on Stat3 and the expression of Stat3-regulated anti-apoptotic genes Bcl-XL, Cryab, Hapa1a, and Mcl1 were higher in the Gprc5a-/- than in Gprc5a+/+ cells. In addition, their responses to Lif were different; Stat3 activation was persistent by Lif treatment in the Gprc5a-/- cells, but was transient in the Gprc5a+/+ cells. The persistent activation of Stat3 by Lif in Gprc5a-/- cells is due to a decreased level of Socs3 protein, a negative inhibitor of the Lif-Stat3 signaling. Restoration of Socs3 inhibited the persistent Stat3 activation in Gprc5a-/- cells. Lung adenocarcinoma cells isolated from Gprc5a-/- mice also exhibited autocrine Lif-mediated Stat3 activation. Treatment of Gprc5a-/- cells isolated from normal and tumor tissue with AG490, a Stat3 signaling inhibitor, or with dominant negative Stat3(Y705F) increased starvation-induced apoptosis and inhibited anchorage-independent growth. These results suggest that persistent Stat3 activation increased the survival and transformation of Gprc5a-/- lung cells. Thus, the tumor suppressive effects of Gprc5a are mediated, at least in part, by inhibition of Stat3 signaling through regulating the stability of the Socs3 protein.

Molecular mechanisms of protein kinase-mediated desensitization and internalization of the $\beta$-adrenergic receptor

The $\beta$-adrenergic receptor ($\beta$AR), which couples to G$\sb{\rm s}$ and activates adenylylcyclase, has been a prototype for studying the activation and desensitization of G-protein-coupled receptors. The main objective of the present study is to elucidate the molecular mechanisms of protein kinase-mediated desensitization and internalization of the $\beta$AR.^ Activation of cAPK or PKC causes a rapid desensitization of $\beta$AR stimulation of adenylylcyclase in L cells, which previous studies suggest involves the cAPK/PKC consensus phosphorylation site in the third intracellular loop of the $\beta$AR, RRSSK$\sp{263}$. To determine the role of the individual serines in the cAPK- and PKC-meditated desensitizations, wild type (WT) and mutant $\beta$ARs containing the substitutions, Ser$\sp{261} \to$ A, Ser$\sp{262} \to$ A, Ser$\sp{262} \to$ D, and Ser$\sp{261/262} \to$ A, were constructed and stably transfected into L cells. The cAPK-mediated desensitization was decreased 70-80% by the Ser$\sp{262} \to$ A, Ser$\sp{262} \to$ D, and the Ser$\sp{261/262} \to$ A mutations, but was not altered by the Ser$\sp{261} \to$ A substitution, demonstrating that Ser$\sp{262}$ was the primary site of the cAPK-induced desensitization. The PMA/PKC-induced desensitization was unaffected by either of the single serine to alanine substitutions, but was reduced 80% by the double serine to alanine substitution, suggesting that either serine was sufficient to confer the PKC-mediated desensitization. Coincident stimulation of cAPK and PKC caused an additive desensitization which was significantly reduced (80%) only by the double substitution mutation. Quantitative evaluation of the coupling efficiencies and the GTP-shift of the WT and mutant receptors demonstrated that only one of the mutants, Ser$\sp{262} \to$ A, was partially uncoupled. The Ser$\sp{262} \to$ D mutation did not significantly uncouple, demonstrating that introducing a negative charge did not appear to mimic the desensitized state of the receptor.^ To accomplish the in vivo phosphorylation of the $\beta$AR, we used two epitope-modified $\beta$ARs, hemagglutinin-tagged $\beta$AR (HA-$\beta$AR) and 6 histidine-tagged $\beta$AR (6His-$\beta$AR), for a high efficiency purification of the $\beta$AR. Neither HA-$\beta$AR nor 6His-$\beta$AR altered activation and desensitization of the $\beta$AR significantly as compared to unmodified wild type $\beta$AR. 61% recovery of ICYP-labeled $\beta$AR was obtained with Ni-NTA column chromatography.^ The truncation 354 mutant $\beta$AR(T354), lacking putative $\beta$ARK site(s), displayed a normal epinephrine stimulation of adenylylcyclase. Although 1.0 $\mu$M epinephrine induced 60% less desensitization in T354 as compared to wild type $\beta$AR, 1.0 $\mu$M epinephrine-mediated desensitization in T354 was 35% greater than PGE$\sb1$-mediated desensitization, which is essentially identical in both WT and T354. These results suggested that sequences downstream of residue 354 may play a role in homologous desensitization and that internalization may be attributed to the additional desensitization besides the cAMP mechanism in T354 $\beta$AR. (Abstract shortened by UMI.) ^

The role of beta-arrestin 2 in lysophosphatidic acid-induced NF-kappaB activation

Lysophosphatidic acid (LPA) is a bioactive phospholipid and binds to its receptors, a family of G protein-coupled receptors (GPCR), which initiates multiple signaling cascades and leads to activation of several transcription factors, including NF-κB. NF-κB critically regulates numerous gene expressions, and is persistently active in many diseases. In our previous studies, we have demonstrated that LPA-induced NF-κB activation is dependent on a novel scaffold protein, CARMA3. However, how CARMA3 is recruited to receptor remains unknown. β-Arrestins are a family of proteins involved in desensitization of GPCR signaling. Additionally, β-arrestins function as signaling adaptor proteins, and mediate multiple signaling pathways. Therefore, we have hypothesized that β-arrestins may link CARMA3 to LPA receptors, and facilitate LPA-induced NF-κB activation. ^ Using β-arrestin-deficient MEFs, we found that β-arrestin 2, but not β-arrestin 1, was required for LPA-induced NF-κB activation. Also, we showed that the expression of NF-κB-dependent cytokines, such as interlukin-6, was impaired in β-arrestin 2-deficient MEFs. Mechanistically, we demonstrated the inducible association of endogenous β-arrestin 2 and CARMA3, and we found the CARD domain of CARMA3 interacted with 60-320 residues of β-arrestin 2. To understand why β-arrestin 2, but not β-arrestin 1, mediated NF-κB activation, we generated β-arrestin mutants. However, some mutants degraded quickly, and the rest did not rescue NF-κB activation in β-arrestin-deficient MEFs, though they had similar binding affinities with CARMA3. Therefore, it indicates that slight changes in residues may determine the different functions of β-arrestins. Moreover, we found β-arrestin 2 deficiency impaired LPA-induced IKK kinase activity, while it did not affect LPA-induced IKKα/β phosphorylation. ^ In summary, our results provide the genetic evidence that β-arrestin 2 serves as a positive regulator in NF-κB signaling pathway by connecting CARMA3 to LPA receptors. Additionally, we demonstrate that β-arrestin 2 is required for IKKα/β activation, but not for the inducible phosphorylation of IKKα/β. Because the signaling pathways around the membrane-proximal region of LPA receptors and GPCRs are quite conserved, our results also suggest a possible link between other GPCRs and CARMA3-mediated NF-κB activation. To fully define the role of β-arrestins in LPA-induced NF-κB signaling pathways will help to identify new drug targets for clinical therapeutics.^

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. ^

Identification of a conserved cluster in the RH domain of GRK critical for activation by GPCRs

One of the most critical aspects of G Protein Coupled Receptors (GPCRs) regulation is their rapid and acute desensitization following agonist stimulation. Phosphorylation of these receptors by GPCR kinases (GRK) is a major mechanism of desensitization. Considerable evidence from studies of rhodopsin kinase and GRK2 suggests there is an allosteric docking site for the receptor distinct from the GRK catalytic site. While the agonist-activated GPCR appears crucial for GRK activation, the molecular details of this interaction remain unclear. Recent studies suggested an important role for the N- and C-termini and domains in the small lobe of the kinase domain in allosteric activation; however, neither the mechanism of action of that site nor the RH domain contributions have been elucidated. To search for the allosteric site, we first indentified evolutionarily conserved sites within the RH and kinase domains presumably deterministic of protein function employing evolutionary trace (ET) methodology and crystal structures of GRK6. Focusing on a conserved cluster centered on helices 3, 9, and 10 in the RH domain, key residues of GRK5 and 6 were targeted for mutagenesis and functional assays. We found that a number of double mutations within helices 3, 9, and 10 and the N-terminus markedly reduced (50–90%) the constitutive phosphorylation of the β-2 Adrenergic Receptor (β2AR) in intact cells and phosphorylation of light-activated rhodopsin (Rho*) in vitro as compared to wild type (WT) GRK5 or 6. Based on these results, we designed peptide mimetics of GRK5 helix 9 both computationally and through chemical modifications with the goal of both confirming the importance of helix 9 and developing a useful inhibitor to disrupt the GPCR-GRK interaction. Several peptides were found to block Rho* phosphorylation by GRK5 including the native helix 9 sequence, Peptide Builder designed-peptide preserving only the key ET residues, and chemically locked helices. Most peptidomimetics showed inhibition of GRK5 activity greater than 80 % with an IC50 of ∼ 30 µM. Alanine scanning of helix 9 has further revealed both essential and non-essential residues for inhibition. Importantly, substitution of Arg 169 by an alanine in the native helix 9-based peptide gave an almost complete inhibition at 30 µM with an IC50 of ∼ 10 µM. In summary we report a previously unrecognized crucial role for the RH domain of GRK5 and 6, and the subsequent identification of a lead peptide inhibitor of protein-protein interaction with potential for specific blockade of GPCR desensitization. ^

Advanced Protein Modeling Method: Benchmarking and Applications in Computer-Aided Drug Discovery

Development of homology modeling methods will remain an area of active research. These methods aim to develop and model increasingly accurate three-dimensional structures of yet uncrystallized therapeutically relevant proteins e.g. Class A G-Protein Coupled Receptors. Incorporating protein flexibility is one way to achieve this goal. Here, I will discuss the enhancement and validation of the ligand-steered modeling, originally developed by Dr. Claudio Cavasotto, via cross modeling of the newly crystallized GPCR structures. This method uses known ligands and known experimental information to optimize relevant protein binding sites by incorporating protein flexibility. The ligand-steered models were able to model, reasonably reproduce binding sites and the co-crystallized native ligand poses of the β2 adrenergic and Adenosine 2A receptors using a single template structure. They also performed better than the choice of template, and crude models in a small scale high-throughput docking experiments and compound selectivity studies. Next, the application of this method to develop high-quality homology models of Cannabinoid Receptor 2, an emerging non-psychotic pain management target, is discussed. These models were validated by their ability to rationalize structure activity relationship data of two, inverse agonist and agonist, series of compounds. The method was also applied to improve the virtual screening performance of the β2 adrenergic crystal structure by optimizing the binding site using β2 specific compounds. These results show the feasibility of optimizing only the pharmacologically relevant protein binding sites and applicability to structure-based drug design projects.

Heterotrimeric G protein -mediated signal transduction in Neurospora crassa

Heterotrimeric G protein-mediated signal transduction is one of numerous means that cells utilize to respond to external stimuli. G proteins consist of α, β andγ subunits. Extracellular ligands bind to seven-transmembrane helix receptors, triggering conformational changes. This is followed by activation of coupled G proteins through the exchange of GDP for GTP on the Gα subunit. Once activated, Gα-GTP dissociates from the βγ dimer. Both of these two moieties can interact with downstream effectors, such as adenylyl cyclase, phospholipase C, phosphodiesterases, or ion channels, leading to a series of changes in cellular metabolism and physiology. ^ Neurospora crassa is a eukaryotic multicellular filamentous fungus, with asexual/vegetative and sexual phases to its life cycle. Three Gα (GNA-1, GNA-2, GNA-3) and one Gβ (GNB-1) proteins have been identified in this organism. This dissertation investigates GNA-1 and GNB-1 mediated signaling pathways in N. crassa. ^ GNA-1 was the first identified microbial Gα that belongs to a mammalian superfamily (Gαi). Deletion of GNA-1 leads to multiple defects in N. crassa. During the asexual cycle, Δgna-1 strains display a slower growth rate and delayed conidiation on solid medium. In the sexual cycle, the Δgna-1 mutant is male-fertile but female-sterile. Biochemical studies have shown that Δ gna-1 strains have lower adenosine 3′–5 ′ cyclic monophosphate (cAMP) levels than wild type under conditions where phenotypic defects are observed. In this thesis work, strains containing one of two GTPase-deficient gna-1 alleles (gna-1 R178C, gna-1Q204L) leading to constitutive activation of GNA-1 have been constructed and characterized. Activation of GNA-1 causes uncontrolled aerial hyphae proliferation, elevated sensitivity to heat and oxidative stresses, and lower carotenoid synthesis. To further study the function of GNA-1, constructs to enable expression of mammalian Gαi superfamily members were transformed into a Δ gna-1 strain, and complementation of Δgna-1 defects investigated. Gαs, which is not a member of Gα i superfamily was used as a control. These mammalian Gα genes were able to rescue the vegetative growth rate defect of the Δ gna-1 strain in the following order: Gαz > Gα o > Gαs > Gαt > Gαi. In contrast, only Gαo was able to complement the sexual defect of a Δgna-1 strain. With regard to the thermotolerance phenotype, none of the mammalian Gα genes restored the sensitivity to a wild type level. These results suggest that GNA-1 regulates two independent pathways during the vegetative and sexual cycles in N. crassa. ^ GNB-1, a G protein β subunit from N. crassa, was identified and its functions investigated in this thesis work. The sequence of the gnb-1 gene predicts a polypeptide of 358 residues with a molecular mass of 39.7 kDa. GNB-1 exhibits 91% identity to Cryphonectria parasitica CPGB-1, and also displays significant homology with human and Dictyostelium Gβ genes (∼66%). A Δ gnb-1 strain was constructed and shown to exhibit defects in asexual spore germination, vacuole number and size, mass accumulation and female fertility. A novel role for GNB-1 in regulation of GNA-1 and GNA-2 protein levels was also demonstrated. ^

The two -state model of receptor activation: The agonist and the efficacy

The Two State model describes how drugs activate receptors by inducing or supporting a conformational change in the receptor from “off” to “on”. The beta 2 adrenergic receptor system is the model system which was used to formalize the concept of two states, and the mechanism of hormone agonist stimulation of this receptor is similar to ligand activation of other seven transmembrane receptors. Hormone binding to beta 2 adrenergic receptors stimulates the intracellular production of cyclic adenosine monophosphate (cAMP), which is mediated through the stimulatory guanyl nucleotide binding protein (Gs) interacting with the membrane bound enzyme adenylylcyclase (AC). ^ The effects of cAMP include protein phosphorylation, metabolic regulation and transcriptional regulation. The beta 2 adrenergic receptor system is the most well known of its family of G protein coupled receptors. Ligands have been scrutinized extensively in search of more effective therapeutic agents at this receptor as well as for insight into the biochemical mechanism of receptor activation. Hormone binding to receptor is thought to induce a conformational change in the receptor that increases its affinity for inactive Gs, catalyzes the release of GDP and subsequent binding of GTP and activation of Gs. ^ However, some beta 2 ligands are more efficient at this transformation than others, and the underlying mechanism for this drug specificity is not fully understood. The central problem in pharmacology is the characterization of drugs in their effect on physiological systems, and consequently, the search for a rational scale of drug effectiveness has been the effort of many investigators, which continues to the present time as models are proposed, tested and modified. ^ The major results of this thesis show that for many b2 -adrenergic ligands, the Two State model is quite adequate to explain their activity, but dobutamine (+/−3,4-dihydroxy-N-[3-(4-hydroxyphenyl)-1-methylpropyl]- b -phenethylamine) fails to conform to the predictions of the Two State model. It is a weak partial agonist, but it forms a large amount of high affinity complexes, and these complexes are formed at low concentrations much better than at higher concentrations. Finally, dobutamine causes the beta 2 adrenergic receptor to form high affinity complexes at a much faster rate than can be accounted for by its low efficiency activating AC. Because the Two State model fails to predict the activity of dobutamine in three different ways, it has been disproven in its strictest form. ^

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.