Acute Regulation of Renal Na+/H+ Exchanger NHE3 by Dopamine: Role of Protein Phosphatase 2A

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56 delta (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, co-immunoprecipitation, blot overlay and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small t antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition. Key words: Natriuresis, Sodium transport, Signal transduction.

Sodium retention and ascites formation in a cholestatic mice model: role of aldosterone and mineralocorticoid receptor?

Renal sodium retention in experimental liver cirrhosis originates from the distal nephron sensitive to aldosterone. The aims of this study were to (1) determine the exact site of sodium retention along the aldosterone-sensitive distal nephron, and (2) to evaluate the role of aldosterone and mineralocorticoid receptor activation in this process. Liver cirrhosis was induced by bile duct ligation in either adrenal-intact or corticosteroid-clamped mice. Corticosteroid-clamp was achieved through adrenalectomy and corticosteroid supplementation with aldosterone and dexamethasone via osmotic minipumps. 24-hours renal sodium balance was evaluated in metabolic cages. Activity and expression of sodium- and potassium-dependent adenosine triphosphatase were determined in microdissected segments of nephron. Within 4-5 weeks, cirrhosis induced sodium retention in adrenal-intact mice and formation of ascites in 50% of mice. At that time, sodium- and potassium-dependent adenosine triphosphatase activity increased specifically in cortical collecting ducts. Hyperaldosteronemia was indicated by increases in urinary aldosterone excretion and in sgk1 (serum- and glucocorticoid-regulated kinase 1) mRNA expression in collecting ducts. Corticosteroid-clamp prevented induction of sgk1 but not cirrhosis-induced sodium retention, formation of ascites and stimulation of sodium- and potassium-dependent adenosine triphosphatase activity and expression (mRNA and protein) in collecting duct. These findings demonstrate that sodium retention in cirrhosis is independent of hyperaldosteronemia and of the activation of mineralocorticoid receptor. CONCLUSION: Bile duct ligation in mice induces cirrhosis which, within 4-5 weeks, leads to the induction of sodium- and potassium-dependent adenosine triphosphatase in cortical collecting ducts, to renal sodium retention and to the formation of ascites. Sodium retention, ascites formation and induction of sodium- and potassium-dependent adenosine triphosphatase are independent of the activation of mineralocorticoid receptors by either aldosterone or glucocorticoids.

Expression of adenosine A2a receptors gene in the olfactory bulb and spinal cord of rat and mouse

The expression of adenosine A2a receptors (A2aR) in the mammalian striatum is well known. In contrast the exact distribution of A2aR in other regions of the central nervous system remains unclear. The aim of this study was to investigate the A2aR gene expression in the rat olfactory bulb and spinal cord, two regions which are seldom included in mapping studies. Secondly, we compared the A2aR expression in the rat and in the mouse brain. Hybridization histochemistry was performed with an S35-labelled radioactive oligonucleotide probe. The results show strong expression of A2aR in the mouse and rat striatum in accordance with previous reports. In the olfactory bulb a weak but specific expression of A2aR was found in the granular cell layer in both species. In contrast, no significant expression of the A2aR gene was observed in other parts of the brain or the rat spinal cord. The presence of the A2aR in the mammalian olfactory bulb suggests a functional role for this receptor in olfaction.

Role of adenosine signaling in penile erection and erectile disorders.

INTRODUCTION: Penile erection is a hemodynamic process, which results from increased flow and retention of blood in the penile organ due to the relaxation of smooth muscle cells. Adenosine, a physiological vasorelaxant, has been shown to be a modulator of penile erection. AIM: To summarize the research on the role of adenosine signaling in normal penile erection and erectile disorders. MAIN OUTCOME MEASURES: Evidence in the literature on the association between adenosine signaling and normal and abnormal penile erection, i.e., erectile dysfunction (ED) and priapism. METHODS: The article reviews the literature on the role of endogenous and exogenous adenosine in normal penile erection, as well as in erectile disorders namely, ED and priapism. RESULTS: Adenosine has been shown to relax corpus cavernosum from various species including human in both in vivo and in vitro studies. Neuromodulatory role of adenosine in corpus cavernosum has also been demonstrated. Impaired adenosine signaling through A(2B) receptor causes partial resistance of corpus cavernosum, from men with organic ED, to adenosine-mediated relaxation. Increased level of adenosine has been shown to be a causative factor for priapism. CONCLUSION: Overall, the research reviewed here suggests a general role of exogenous and endogenous adenosine signaling in normal penile erection. From this perspective, it is not surprising that impaired adenosine signaling is associated with ED, and excessive adenosine signaling is associated with priapism. Adenosine signaling represents a potentially important diagnostic and therapeutic target for the treatment of ED and priapism.

Adenosine signaling contributes to ethanol-induced fatty liver in mice.

Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5'-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5'-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver.

CD73-generated adenosine restricts lymphocyte migration into draining lymph nodes.

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5'-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73(-/-) mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73(+/+) and cd73(-/-) lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A(2B) receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-alpha. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73(-/-) mice is largely normalized by pretreatment with the selective A(2B) receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.

Enterococcus faecalis PcfC, a spatially localized substrate receptor for type IV secretion of the pCF10 transfer intermediate.

Upon sensing of peptide pheromone, Enterococcus faecalis efficiently transfers plasmid pCF10 through a type IV secretion (T4S) system to recipient cells. The PcfF accessory factor and PcfG relaxase initiate transfer by catalyzing strand-specific nicking at the pCF10 origin of transfer sequence (oriT). Here, we present evidence that PcfF and PcfG spatially coordinate docking of the pCF10 transfer intermediate with PcfC, a membrane-bound putative ATPase related to the coupling proteins of gram-negative T4S machines. PcfC and PcfG fractionated with the membrane and PcfF with the cytoplasm, yet all three proteins formed several punctate foci at the peripheries of pheromone-induced cells as monitored by immunofluorescence microscopy. A PcfC Walker A nucleoside triphosphate (NTP) binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCDelta N103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCDelta N103 bound pCF10 but not pcfG or Delta oriT mutant plasmids as shown by transfer DNA immunoprecipitation, indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCDelta N103 bound DNA substrates and interacted with purified PcfF and PcfG in vitro. Our findings support a model in which (i) PcfF recruits PcfG to oriT to catalyze T-strand nicking, (ii) PcfF and PcfG spatially position the relaxosome at the cell membrane to stimulate substrate docking with PcfC, and (iii) PcfC initiates substrate transfer through the pCF10 T4S channel by an NTP-dependent mechanism.

The Role of IL-6 in Adenosine-Mediated Pulmonary Fibrosis

Adenosine is a purinergic signaling molecule that regulates various aspects of inflammation and has been implicated in the pathogenesis of chronic lung diseases. Previous studies have demonstrated that adenosine up-regulates IL-6 production through the engagement of the A2B adenosine receptor in various cell types, including alveolar macrophages. IL-6 is elevated in mouse models and humans with chronic lung disease, suggesting a potential role in disease progression. Furthermore, chronic elevation of adenosine in the lungs of adenosine deaminase deficient (Ada-/-) mice leads to the development of pulmonary inflammation, alveolar destruction, and fibrosis, in conjunction with IL-6 elevation. Thus, it was hypothesized that IL-6 contributes to pulmonary inflammation and fibrosis in this model. To test this hypothesis, Ada/IL-6 double knockout mice (Ada/IL-6-/-) were generated to assess the consequences of genetically removing IL-6 on adenosine-dependent pulmonary injury. Ada/IL-6-/- mice exhibited a significant reduction in inflammation, alveolar destruction, and pulmonary fibrosis. Next, Ada-/- mice were treated systematically with IL-6 neutralizing antibodies to test the efficacy of blocking IL-6 on chronic lung disease. These treatments were associated with decreased pulmonary inflammation, alveolar destruction, and fibrosis. To determine the role of IL-6 in a second model of pulmonary fibrosis, wild type mice and IL-6-/- mice were subjected to intraperitoneal injections of bleomycin twice a week for four weeks. Results demonstrated that IL-6-/- mice developed reduced pulmonary fibrosis. To examine a therapeutic approach in this model, wild type mice exposed to bleomycin were treated with IL-6 neutralizing antibodies. Similar results were observed as with Ada-/- mice, namely diminished pulmonary inflammation and fibrosis. In both models, elevations in IL-6 were associated with increased phosphorylated STAT-3 in the nuclei of numerous cell types in the airways, including type II alveolar epithelial cells (AEC). Genetic removal and neutralization of IL-6 in both models was associated with decreased STAT-3 activation in type II AEC. The mechanism of activation in these cells that lack the membrane bound IL-6Ra suggests IL-6 trans-signaling may play a role in regulating fibrosis. Characterization of this mechanism demonstrated that the soluble IL-6Ra (sIL-6Ra) is upregulated in both models during chronic conditions. In vitro studies in MLE-12 alveolar epithelial cells confirmed that IL-6, in combination with the sIL-6Ra, activates STAT-3 and TWIST in association with enhancement of epithelial-to-mesenchymal transition, which can contribute to fibrosis. Similarly, patients with idiopathic pulmonary fibrosis demonstrated a similar pattern of increased IL-6 expression, STAT-3 activation, and sIL-6Ra increases. These findings demonstrate that adenosine-dependent elevations in IL-6 contribute to the development and progression of pulmonary inflammation and fibrosis. The implications from these studies are that adenosine and/or IL-6 neutralizing agents represent novel therapeutic targets for the treatment of pulmonary disorders where fibrosis is a detrimental component.

Promotion of liver regeneration by natural killer cells in a murine model is dependent on extracellular adenosine triphosphate phosphohydrolysis

Nucleotides, such as adenosine triphosphate (ATP), are released by cellular injury, bind to purinergic receptors expressed on hepatic parenchymal and nonparenchymal cells, and modulate cellular crosstalk. Liver resection and resulting cellular stress initiate such purinergic signaling responses between hepatocytes and innate immune cells, which regulate and ultimately drive liver regeneration. We studied a murine model of partial hepatectomy using immunodeficient mice to determine the effects of natural killer (NK) cell-mediated purinergic signaling on liver regeneration. We noted first that liver NK cells undergo phenotypic changes post-partial hepatectomy (PH) in vivo, including increased cytotoxicity and more immature phenotype manifested by alterations in the expression of CD107a, CD27, CD11b, and CD16. Hepatocellular proliferation is significantly decreased in Rag2/common gamma-null mice (lacking T, B, and NK cells) when compared to wildtype and Rag1-null mice (lacking T and B cells but retaining NK cells). Extracellular ATP levels are elevated post-PH and NK cell cytotoxicity is substantively increased in vivo in response to hydrolysis of extracellular ATP levels by apyrase (soluble NTPDase). Moreover, liver regeneration is significantly increased by the scavenging of extracellular ATP in wildtype mice and in Rag2/common gamma-null mice after adoptive transfer of NK cells. Blockade of NKG2D-dependent interactions significantly decreased hepatocellular proliferation. In vitro, NK cell cytotoxicity is inhibited by extracellular ATP in a manner dependent on P2Y1, P2Y2, and P2X3 receptor activation. Conclusion: We propose that hepatic NK cells are activated and cytotoxic post-PH and support hepatocellular proliferation. NK cell cytotoxicity is, however, attenuated by hepatic release of extracellular ATP by way of the activation of specific P2 receptors. Clearance of extracellular ATP elevates NK cell cytotoxicity and boosts liver regeneration.

Development of small molecular tools for the cellular study of adenosine receptors

The adenosine receptors are members of the G-protein coupled receptor (GPCR) family which represents the largest class of cell-surface proteins mediating cellular communication. As a result, GPCRs are formidable drug targets and it is estimated that approximately 30% of the marketed drugs act through members of this receptor class. There are four known subtypes of adenosine receptors: A1, A2A, A2B and A3. The adenosine A1 receptor, which is the subject of this presentation, mediates the physiological effects of adenosine in various tissues including the brain, heart, kidney and adipocytes. In the brain for instance, its role in epilepsy and ischemia has been the focus of many studies. Previous attempts to study the biosynthesis, trafficking and agonist-induced internalisation of the adenosine A1 receptor in neurons using fluorescent protein-receptor fusion constructs have been hampered by the sheer size of the fluorescent protein (GFP) that ultimately affected the function of the receptor. We have therefore initiated a research programme to develop small molecule fluorescent agonists that selectively activate the adenosine A1 receptor. Our probe design is based on the endogenous ligand adenosine and the known unselective adenosine receptor agonist NECA. We have synthesised a small library of non-fluorescent adenosine derivatives that have different cyclic and bicyclic moieties at the 6 position of the purine ring and have evaluated the pharmacology of these compounds using a yeast-based assay. This analysis revealed compounds with interesting behaviour, i.e. exhibiting subtype-selectivity and biased signalling, that can be potentially used as tool compounds in their own right for cellular studies of the adenosine A1 receptor. Furthermore, we have also linked fluorescent dyes to the purine ring and discovered fluorescent compounds that can activate the adenosine A1 receptor.

REGULATION OF BRAIN NORADRENERGIC-RECEPTOR FUNCTION BY ADRENOCORTICOTROPHIN AND ANTIDEPRESSANT DRUGS (ADRENERGIC RECEPTOR, CYCLIC-AMP, ADENYLATE CYCLASE, IMIPRAMINE, STRESS)

Human behavior appears to be regulated in part by noradrenergic transmission since antidepressant drugs modify the number and function of (beta)-adrenergic receptors in the central nervous system. Affective illness is also known to be associated with the endocrine system, particularly the hypothalamic-pituitary-adrenal axis. The aim of the present study was to determine whether hormones, in particular adrencorticotrophin (ACTH) and corticosterone, may influence behavior by regulating brain noradrenergic receptor function.^ Chronic treatment with ACTH accelerated the increase or decrease in rat brain (beta)-adrenergic receptor number induced by a lesion of the dorsal noradrenergic bundle or treatment with the antidepressant imipramine. Chronic administration of ACTH alone had no effect on (beta)-receptor number although it reduced norepinephrine stimulated cyclic AMP accumulation in brain slices. Treatment with imipramine also reduced the cyclic AMP response to norepinephrine but was accompanied by a decrease in (beta)-adrenergic receptor number. Both the imipramine and ACTH treatments reduced the affinity of (beta)-adrenergic receptors for norepinephrine, but only the antidepressant modified the potency of the neurotransmitter to stimulate second messenger production. Neither ACTH nor imipramine treatment altered Gpp(NH)p- or fluoride-stimulated adenylate cyclase, cyclic AMP, cyclic GMP, or cyclic GMP-stimulated cyclic AMP phosphodiesterase, or the activity of the guanine nucleotide binding protein (Gs). These findings suggested that post-receptor components of the cyclic nucleotide generating system are not influenced by the hormone or antidepressant. This conclusion was verified by the finding that neither treatment altered adenosine-stimulated cyclic AMP accumulation in brain tissue.^ A detailed examination of the (alpha)- and (beta)-adrenergic receptor components of norepinephrine-stimulated cyclic AMP production revealed that ACTH, but not imipramine, administration reduced the contribution of the (alpha)-receptor mediated response. Like ACTH treatment, corticosterone diminished the (alpha)-adrenergic component indicating that adrenal steroids probably mediate the neurochemical responses to ACTH administration. The data indicate that adrenal steroids and antidepressants decrease noradrenergic receptor function by selectively modifying the (alpha)- and (beta)-receptor components. The functional similarity in the action of the steroid and antidepressants suggests that adrenal hormones normally contribute to the maintenance of receptor systems which regulate affective behavior in man. ^

Potential Role of Adenosine Signaling in Acetic Acid Activation of Murine Sensory Neurons

Chronic respiratory illnesses are a significant cause of morbidity and mortality, and acute changes in respiratory function often lead to hospitalization. Air pollution is known to exacerbate asthma, but the molecular mechanisms of this are poorly understood. The current studies were aimed at clarifying the roles of nerve subtypes and purinergic receptors in respiratory reflex responses following exposure to irritants. In C57Bl/6J female mice, inspired adenosine produced sensory irritation, shown to be mediated mostly by A-delta fibers. Secondly, the response to inhaled acetic acid was discovered to be dually influenced by C and A-delta fibers, as indicated by the observed effects of capsaicin pretreatment, which selectively destroys TRPV1-expressing fibers (mostly C fibers) and pretreatment with theophylline, a nonselective adenosine receptor antagonist. The responses to both adenosine and acetic acid were enhanced in the ovalbumin-allergic airway disease model, although the particular pathway altered is still unknown.

The role of adenosine in pulmonary angiogenesis

Angiogenesis is a feature of chronic lung diseases such as asthma and pulmonary fibrosis; however, the pathways controlling pathological angiogenesis during lung disease are not completely understood. Adenosine is a signaling nucleoside that accumulates as a result of tissue hypoxia and damage. Adenosine has been implicated in the exacerbation of chronic lung disease and in the regulation of angiogenesis; however, the relationship between these factors has not been investigated. The work presented in this dissertation utilized adenosine deaminase (ADA)-deficient mice to determine whether chronic elevations of adenosine in vivo result in pulmonary angiogenesis, and to identify factors that could potentially mediate this process. Results demonstrate that there is substantial angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. Replacement enzyme therapy with pegylated ADA resulted in a lowering of adenosine levels and reversal of tracheal angiogenesis, indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the ELR+ angiogenic chemokine CXCL1 were found to be elevated in an adenosine-dependent manner in the lungs of ADA-deficient mice. Neutralization of CXCL1 and its putative receptor, CXCR2, in ADA-deficient lung lysates resulted in the inhibition of angiogenic activity suggesting that CXCL1 signaling through the CXCR2 receptor is responsible for mediating the observed increases in angiogenesis. Taken together, these findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis and may therefore represent an important therapeutic target for the treatment of pathological angiogenesis. ^

New insights into the role of the duodenal vitamin D receptor in calcium homeostasis

It is generally believed that 1,25(OH)2D3, bound to its receptor (VDR) contributes to calcium homeostasis by regulating active calcium absorption in the proximal small intestine. However, studying patients with hereditary vitamin D-resistant rickets (HVDRR) provided investigators with a better understanding of VDR's role in calcium homeostasis. HVDRR patients have inactivating mutations in the VDR, and as a consequence they develop hypocalcemia, hyperparathyroidism and severe rickets. However, these phenotypes can be corrected if the patients are given IV infusions of calcium or dietary calcium. This raises the question of what is the physiological significance of VDR-regulated active calcium absorption if calcium homeostasis can be restored independently of the VDR. ^ In order to distinguish the contribution of VDR in the proximal small intestine to overall calcium homeostasis, I generated transgenic mice expressing the human VDR (hVDR) exclusively in the proximal small intestine of mVDR-/- mice by using an hVDR-expressing transgene driven by the duodenal-specific adenosine deaminase enhancer (hVDR+/mVDR-/-). hVDR+/mVDR-/- mice expressed transcriptionally active hVDR only in the proximal small intestine and responded to 1,25(OH)2D3 by up-regulating expression of TRPV6 and calbindin D9K, genes involved in calcium absorption. Furthermore, ligated duodenal loop assays determined that calcium absorption in hVDR+/mVDR-/- mice was as responsive to 1,25(OH)2D3 as in WT mice. Despite having a functional hVDR in the proximal small intestine, hVDR+/mVDR-/- mice were hypocalcemic, had hyperparathyroidism, and were rachitic when fed a normal rodent diet at weaning, as were the mVDR-/- mice. However, when fed a high calcium, phosphorus, and lactose diet (rescue diet), the hVDR+/mVDR-/- mice responded more effectively than the mVDR-/- mice by down-regulation of parathyroid hormone production and by a greater increase in bone mineralization. Furthermore, when three-month-old rachitic mice were fed a rescue diet for 3 weeks, serum calcium and bone mineral content were normalized in hVDR+/mVDR-/- mice, but not in mVDR-/- mice. ^ In conclusion, hVDR expression enabled young mice to better use the rescue diet than mVDR-/- mice. Expression of transgenic hVDR also protected the ability of older mice to respond to the rescue diet despite the absence of the VDR elsewhere in the intestinal tract. I propose that because hVDR+/mVDR-/- mice responded better than mVDR-/- mice to the rescue diet, it is likely that VDR expression in the proximal small intestine is necessary in nutritional (insufficient dietary calcium) and physiological (age) conditions when passive calcium absorption is inadequate. ^

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.