Endogenous regulator of G protein signaling proteins suppress Galphao-dependent, mu-opioid agonist-mediated adenylyl cyclase supersensitization

Chronic mu-opioid agonist treatment leads to dependence with withdrawal on removal of agonist. At the cellular level withdrawal is accompanied by a supersensitization of adenylyl cyclase, an effect that requires inhibitory Galpha proteins. Inhibitory Galpha protein action is modulated by regulator of G protein signaling (RGS) proteins that act as GTPase activating proteins and reduce the lifetime of Galpha-GTP. In this article, we use C6 glioma cells expressing the rat mu-opioid receptor (C6mu) to examine the hypothesis that Galphao alone can mediate mu-opioid agonist induced adenylyl cyclase supersensitivity and that endogenous RGS proteins serve to limit the extent of this supersensitization. C6mu cells were stably transfected with pertussis toxin (PTX)-insensitive Galphao that was either sensitive or insensitive to endogenous RGS proteins. Cells were treated with PTX to uncouple endogenous Galpha proteins followed by exposure to the mu-opioid agonists [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin or morphine. Supersensitization was observed in cells expressing wild-type Galpha, but this was lost on PTX treatment. In cells expressing PTX-insensitive Galphao supersensitization was recovered, confirming that Galphao alone can support supersensitization. In cells expressing the RGS-insensitive mutant Galphao, there was a greater degree of supersensitization and the concentration of micro-agonist needed to achieve half-maximal supersensitization was reduced by 10-fold. The amount of supersensitization seen did not directly relate to the degree of acute inhibition of adenylyl cyclase. These results demonstrate a role for Galphao in adenylyl cyclase supersensitization after mu-agonist exposure and show that this action is modulated by endogenous RGS proteins.     

Dissociation of mu opioid tolerance from receptor down-regulation in rat spinal cord

The effect of continuous intrathecal infusions of opioids was studied in rats. Chronic intrathecal infusion of the highly selective mu agonist, [NMPhe3, D-Pro4]morphiceptin produced a rapid onset of tolerance to the drug in the analgesic test. However, membrane prepared from the spinal cords of the rats chronically infused with a low dose of the drug showed no statistically significant change in the number of mu or delta receptor binding sites. In addition, membrane prepared from rats challenged with a single high-dose bolus injection of [NMPhe3, D-Pro4]morphiceptin did not produce alterations in the receptor binding number. If the chronically treated rats were challenged with an acute bolus dose of [NMPhe3, D-Pro4]morphiceptin, there was a significant decrease in the number of binding sites. The reduced binding site number was observed for the mu ligand but not for the delta ligand. A similar decrease of receptor binding can also be achieved by chronic infusion of the drug at high doses. Scatchard plot showed a decrease of maximum mu binding sites in the membranes prepared from the combined chronic infusion-acute injection treated rats. Brain tissue from the same rats showed no change in the number of mu and delta receptor binding sites, indicating that the down-regulation of mu receptors was confined to the spinal cord only. Morphine did not induce receptor down-regulation by acute, chronic or combined treatments. These results suggest that in the rat spinal cord, tolerance can be induced without apparent receptor down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of agonists on adenylyl cyclase superactivation mediated by the kappa opioid receptors: adenylyl cyclase superactivation is independent of agonist-induced phosphorylation, desensitization, internalization, and down-regulation

Prolonged activation of opioid receptors followed by agonist removal leads to adenylyl cyclase (AC) superactivation. In this study, we examined in CHO cells stably expressing the human or rat kappa opioid receptor (hkor or rkor) whether agonists had differential abilities to induce AC superactivation and whether the hkor and rkor exhibited differential AC superactivation. Pretreatment of the hkor with (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate (U50,488H) induced AC superactivation in a time- and dose-dependent manner, reaching a plateau at 4 h and 0.1 microM. The extents of AC superactivation after a 4-h pretreatment of the hkor with saturating concentrations of agonists were in the order of the full agonists U50,488H, dynorphin A(1-17), (+/-)-ethylketocyclazocine, etorphine, and U69,593 > the high-efficacy partial agonist nalorphine > the low-efficacy partial agonists nalbuphine, morphine, and pentazocine. Interestingly, the full agonist levorphanol caused much lower AC superactivation than other full agonists and reduced the AC superactivation induced by U50,488H and dynorphin A(1-17) in a dose-dependent manner. The order of relative efficacies of agonists in causing AC superactivation mediated by the rkor was similar to that mediated by the hkor and the extents of AC superactivation were slightly lower. Because the rkor does not undergo U50,488H (1 microM)-induced phosphorylation, desensitization, internalization, and down-regulation in these cells, the degree of AC superactivation is independent of these processes. This is among the first reports to demonstrate that relative efficacies of agonists in causing AC superactivation generally correlated with those in activating G proteins and a full agonist reduced AC superactivation induced by another full agonist.     

LPS-induced down-regulation of signal regulatory protein {alpha} contributes to innate immune activation in macrophages

Activation of the mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) cascades after Toll-like receptor (TLR) stimulation contributes to innate immune responses. Signal regulatory protein (SIRP) α, a member of the SIRP family that is abundantly expressed in macrophages, has been implicated in regulating MAPK and NF-κB signaling pathways. In addition, SIRPα can negatively regulate the phagocytosis of host cells by macrophages, indicating an inhibitory role of SIRPα in innate immunity. We provide evidences that SIRPα is an essential endogenous regulator of the innate immune activation upon lipopolysaccharide (LPS) exposure. SIRPα expression was promptly reduced in macrophages after LPS stimulation. The decrease in SIRPα expression levels was required for initiation of LPS-induced innate immune responses because overexpression of SIRPα reduced macrophage responses to LPS. Knockdown of SIRPα caused prolonged activation of MAPKs and NF-κB pathways and augmented production of proinflammatory cytokines and type I interferon (IFN). Mice transferred with SIRPα-depleted macrophages were highly susceptible to endotoxic shock, developing multiple organ failure and exhibiting a remarkable increase in mortality. SIRPα may accomplish this mainly through its association and sequestration of the LPS signal transducer SHP-2. Thus, SIRPα functions as a biologically important modulator of TLR signaling and innate immunity.     

ATP receptor regulation of adenylate cyclase and protein kinase C activity in cultured renal LLC-PK1 cells.

In cultured intact LLC-PK1 renal epithelial cells, a nonhydrolyzable ATP analogue, ATP gamma S, inhibits AVP-stimulated cAMP formation. In LLC-PK1 membranes, several ATP analogues inhibit basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in a dose-dependent manner. The rank order potency of inhibition by ATP analogues suggests that a P2y type of ATP receptor is involved in this inhibition. The compound ATP gamma S inhibits agonist-stimulated adenylate cyclase activity in solubilized and in isobutylmethylxanthine (IBMX) and quinacrine pretreated membranes, suggesting that ATP gamma S inhibition occurs independent of AVP and A1 adenosine receptors and of phospholipase A2 activity. The ATP gamma S inhibition of AVP-stimulated adenylate cyclase activity is not affected by pertussis toxin but is attenuated by GDP beta S, suggesting a possible role for a pertussis toxin insensitive G protein in the inhibition. Exposure of intact LLC-PK cells to ATP gamma S results in a significant increase in protein kinase C activity. However, neither of two protein kinase C inhibitors (staurosporine and H-7) prevents ATP gamma S inhibition of AVP-stimulated adenylate cyclase activity, suggesting that this inhibition occurs by a protein kinase C independent mechanism. These findings suggest the presence of functional P2y purinoceptors coupled to two signal transduction pathways in cultured renal epithelial cells. The effect of P2y purinoceptors to inhibit AVP-stimulated adenylate cyclase activity may be mediated, at least in part, by a pertussis toxin insensitive G protein.     

Efficacy and tolerance of narcotic analgesics at the mu opioid receptor in differentiated human neuroblastoma cells

Upon differentiation with retinoic acid of the human neuroblastoma cells SH-SY5Y into mature neurons, opioid drugs become highly effective in suppressing prostaglandin E1 (50% inhibition)- and forskolin (70% inhibition)-stimulated adenylate cyclase activity, which was assessed by measuring cyclic AMP accumulation in intact cells. Whereas the SH-SY5Y cells carry both mu and delta receptors in a ratio of mu/delta approximately equal to 5/1, the response is predominantly mediated by the mu receptor. Morphine acts as a strong agonist with an EC50 of 50 to 100 nM which falls into the therapeutic range expected for narcotic analgesic effects mediated by the mu receptor. Narcotic analgesic drugs with only partial agonism fail to evoke full response, which suggests that this cell model could provide a rapid screening assay for narcotic analgesic efficacy. Continued exposure of the cells to morphine resulted in partial tolerance within 12 hr with a 4-fold shift of morphine's EC50 to higher concentrations, whereas longer morphine exposure did not cause any further shift. Thus, the differentiated SH-SY5Y cells provide a suitable system for studying the molecular mechanisms of the narcotic analgesics.     

Orphanin FQ/nociceptin-mediated desensitization of opioid receptor-like 1 receptor and mu opioid receptors involves protein kinase C: a molecular mechanism for heterologous cross-talk

Morphine tolerance in vivo is reduced following blockade of the orphanin FQ/nociceptin (OFQ/N)/opioid receptor-like 1 (ORL1) receptor system, suggesting that OFQ/N contributes to the development of morphine tolerance. We previously reported that a 60-min activation of ORL1 receptors natively expressed in BE(2)-C cells desensitized both mu and ORL1 receptor-mediated inhibition of cAMP. Investigating the mechanism(s) of OFQ/N-mediated mu and ORL1 receptor cross-talk, we found that pretreatment with the protein kinase C inhibitor, chelerythrine chloride (1 microM), blocked OFQ/N-mediated homologous desensitization of ORL1 and heterologous desensitization of mu opioid receptors. Furthermore, depletion of PKC by 12-O-tetradecanoylphorbol-13-acetate exposure (48 h, 1 microM) also prevented OFQ/N-mediated mu and ORL1 desensitization. OFQ/N pretreatment resulted in translocation of PKC-alpha, G protein-coupled receptor kinase 2 (GRK2) and GRK3 from the cytosol to the membrane, and this translocation was also blocked by chelerythrine. Reduction of GRK2 and GRK3 levels by antisense, but not sense DNA treatment blocks ORL1 and mu receptor desensitization. This suggests that PKC-alpha is required for GRK2 and GRK3 translocation to the membrane, where GRK can inactivate ORL1 and mu opioid receptors upon rechallenge with the appropriate agonist. Our results demonstrate for the first time the involvement of conventional PKC isozymes in OFQ/N-induced mu-ORL1 cross-talk, and represent a possible mechanism for OFQ/N-induced anti-opioid actions.     

5-Aminoimidazole-4-carboxamide riboside sensitizes TRAIL- and TNF{alpha}-induced cytotoxicity in colon cancer cells through AMP-activated protein kinase signaling

Death receptor-mediated tumor cell death, either alone or in combination with other anticancer drugs, is considered as a new strategy for anticancer therapy. In this study, we have investigated the effects and molecular mechanisms of 5-aminoimidazole-4-carboxamide riboside [AICAR; a pharmacologic activator of AMP-activated protein kinase (AMPK)] in sensitizing tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)- and TNFalpha-induced apoptosis of human colon cancer HCT116 cells. The cytotoxic action of AICAR requires AMPK activation and may occur at various stages of apoptotic pathways. AICAR cotreatment with either TRAIL or TNFalpha enhances activities of caspase-8, caspase-9, and caspase-3; down-regulates the antiapoptotic protein Bcl-2; increases the cleavage of Bid and results in the decrease of mitochondrial membrane potential; potentiates activation of p38 and c-Jun NH(2)-terminal kinase; and inhibits nuclear factor-kappaB activity. In addition, this sensitized cell apoptosis was neither observed in p53-null HCT116 cells nor affected by the cotreatment with mevalonate. In summary, we have developed a novel strategy of combining AICAR with TRAIL for the treatment of colon cancer cells. The sensitization effect of AICAR in cell apoptosis was mediated through AMPK pathway, requires p53 activity, and involves mitochondria-dependent apoptotic cascades, p38 and c-Jun NH(2)-terminal kinase.     

The role of C4-binding protein and {beta }1H in proteolysis of C4b and C3b

Two forms of C4-binding protein (C4-bp) (C4-bp low, C4-bp high), which differ slightly in net charge and apparent molecular weight, as determined by SDS- PAGE, were separated by ion-exchange chromatography and contaminants removed with specific antisera. Both forms of C4-bp served as cofactors for the cleavage of C4b in solution by C3b inactivator, and the resulting fragments of the a’-chain of C4b had identical molecular weights. In addition, similarly to β1H, C4-bp low or high served as cofactors for the cleavage of fluid phase C3b by C3bINA. However, important quantitative differences between the activities of C4-bp and β1H were observed. With regard to C3b in solution, the cofactor activity of β1H was {approximately equal to}20 times greater than that of C4-bp on a weight basis. In relation to cell-bound C3b, the differences in activity were even more marked. Whereas β1H enhanced the effects of C3bINA on the erythrocyte intermediate EC3b, inhibiting the assembly of EC3bBb, C4-bp was without effect even at concentrations {approximately equal to}300 times greater than β1H. Therefore, under physiological conditions, it is likely that β1H is the key protein which controls the function of C3b, and that C4-bp activity is directed mainly toward the cleavage of C4b. We also examined the relation between C4-bp and the C3b-C4bINA cofactor described by Stroud and collaborators (3, 4). By functional, physico-chemical and immunological criteria, they are the same protein.     

Exisulind and guanylyl cyclase C induce distinct antineoplastic signaling mechanisms in human colon cancer cells

The nonsteroidal anti-inflammatory drug sulindac is metabolized to sulindac sulfone (exisulind), an antineoplastic agent that inhibits growth and induces apoptosis in solid tumors. In colon cancer cells, the antineoplastic effects of exisulind have been attributed, in part, to induction of cyclic guanosine 3',5'-monophosphate (cGMP) signaling through inhibition of cGMP-specific phosphodiesterases, which elevates intracellular cGMP, and novel expression of cGMP-dependent protein kinase (PKG) Ibeta, the presumed downstream effector mediating apoptosis. Here, inhibition of proliferation and induction of cell death by exisulind was dissociated from cGMP signaling in human colon cancer cells. Accumulation of intracellular cGMP produced by an exogenous cell-permeant analogue of cGMP or a potent agonist of guanylyl cyclase C yielded cytostasis without cell death. Surprisingly, the antiproliferative effects of induced cGMP accumulation were paradoxically less than additive, rather than synergistic, when combined with exisulind. Further, although exisulind induced expression of PKG Ibeta, it did not elevate intracellular cGMP and its efficacy was not altered by inhibition or activation of PKG I. Rather, PKG I induced by exisulind may mediate desensitization of cytostasis induced by cGMP. Thus, cytotoxic effects of exisulind are independent of cGMP signaling in human colon cancer cells. Moreover, combination therapies, including exisulind and agents that induce cGMP signaling, may require careful evaluation in patients with colon cancer.     

The origins of diversity and specificity in g protein-coupled receptor signaling

The modulation of transmembrane signaling by G protein-coupled receptors (GPCRs) constitutes the single most important therapeutic target in medicine. Drugs acting on GPCRs have traditionally been classified as agonists, partial agonists, or antagonists based on a two-state model of receptor function embodied in the ternary complex model. Over the past decade, however, many lines of investigation have shown that GPCR signaling exhibits greater diversity and "texture" than previously appreciated. Signal diversity arises from numerous factors, among which are the ability of receptors to adopt multiple "active" states with different effector-coupling profiles; the formation of receptor dimers that exhibit unique pharmacology, signaling, and trafficking; the dissociation of receptor "activation" from desensitization and internalization; and the discovery that non-G protein effectors mediate some aspects of GPCR signaling. At the same time, clustering of GPCRs with their downstream effectors in membrane microdomains and interactions between receptors and a plethora of multidomain scaffolding proteins and accessory/chaperone molecules confer signal preorganization, efficiency, and specificity. In this context, the concept of agonist-selective trafficking of receptor signaling, which recognizes that a bound ligand may select between a menu of active receptor conformations and induce only a subset of the possible response profile, presents the opportunity to develop drugs that change the quality as well as the quantity of efficacy. As a more comprehensive understanding of the complexity of GPCR signaling is developed, the rational design of ligands possessing increased specific efficacy and attenuated side effects may become the standard mode of drug development.     

Dynamics of {beta}-amyloid reductions in brain, cerebrospinal fluid, and plasma of {beta}-amyloid precursor protein transgenic mice treated with a {gamma}-secretase inhibitor

gamma-Secretase inhibitors are one promising approach to the development of a therapeutic for Alzheimer's disease (AD). gamma-Secretase inhibitors reduce brain beta-amyloid peptide (Abeta), which is believed to be a major contributor in the etiology of AD. Transgenic mice overexpressing the human beta-amyloid precursor protein (APP) are valuable models to examine the dynamics of Abeta changes with gamma-secretase inhibitors in plaque-free and plaque-bearing animals. BMS-299897 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid, a gamma-secretase inhibitor, showed dose- and time dependent reductions of Abeta in brain, cerebrospinal fluid (CSF), and plasma in young transgenic mice, with a significant correlation between brain and CSF Abeta levels. Because CSF and brain interstitial fluid are distinct compartments in composition and location, this correlation could not be assumed. In contrast, aged transgenic mice with large accumulations of Abeta in plaques showed reductions in CSF Abeta in the absence of measurable changes in plaque Abeta in the brain after up to 2 weeks of treatment. Hence, CSF Abeta levels were a valuable measure of gamma-secretase activity in the central nervous system in either the presence or absence of plaques. Transgenic mice were also used to examine potential side effects due to Notch inhibition. BMS-299897 was 15-fold more effective at preventing the cleavage of APP than of Notch in vitro. No changes in the maturation of CD8(+) thymocytes or of intestinal goblet cells were observed in mice treated with BMS-299897, showing that it is possible for gamma-secretase inhibitors to reduce brain Abeta without causing Notch-mediated toxicity.     

Identification of an alternative G{alpha}q-dependent chemokine receptor signal transduction pathway in dendritic cells and granulocytes

CD38 controls the chemotaxis of leukocytes to some, but not all, chemokines, suggesting that chemokine receptor signaling in leukocytes is more diverse than previously appreciated. To determine the basis for this signaling heterogeneity, we examined the chemokine receptors that signal in a CD38-dependent manner and identified a novel "alternative" chemokine receptor signaling pathway. Similar to the "classical" signaling pathway, the alternative chemokine receptor pathway is activated by Galpha(i2)-containing Gi proteins. However, unlike the classical pathway, the alternative pathway is also dependent on the Gq class of G proteins. We show that Galpha(q)-deficient neutrophils and dendritic cells (DCs) make defective calcium and chemotactic responses upon stimulation with N-formyl methionyl leucyl phenylalanine and CC chemokine ligand (CCL) 3 (neutrophils), or upon stimulation with CCL2, CCL19, CCL21, and CXC chemokine ligand (CXCL) 12 (DCs). In contrast, Galpha(q)-deficient T cell responses to CXCL12 and CCL19 remain intact. Thus, the alternative chemokine receptor pathway controls the migration of only a subset of cells. Regardless, the novel alternative chemokine receptor signaling pathway appears to be critically important for the initiation of inflammatory responses, as Galpha(q) is required for the migration of DCs from the skin to draining lymph nodes after fluorescein isothiocyanate sensitization and the emigration of monocytes from the bone marrow into inflamed skin after contact sensitization.     

Specific hammerhead ribozymes reduce synthesis of cation-independent mannose 6-phosphate receptor mRNA and protein

Storage diseases because of lysosomal enzyme deficiencies may be treated by the transplantation of cells that secrete the enzyme which is deficient in patients. One can expect that increasing the amount of secreted enzymes will improve the therapy efficacy. Secretion of lysosomal enzymes can be enhanced by reducing the mannose 6-phosphate receptor involved in the lysosomal sorting of newly synthesized lysosomal enzymes. For this purpose, we have constructed hammerhead ribozymes targeting the mRNA of the large murine mannose 6-phosphate receptor (M6PR300). In vitro ribozymes cleave M6PR300 RNA fragments efficiently with cleavage rates of 69-93% after 3 h of incubation. Ribozymes were cloned into an expression vector in which they are integrated into the VaI adenovirus RNA to increase stability and in which they are transcribed from an RNA polymerase III promoter. These plasmids were transiently transfected into BHK cells to investigate in vivo activity. Two ribozymes reduce efficiently the levels of murine M6PR300 mRNA in transient transfection experiments to 42-45%. This correlates with the reduction of M6PR300 biosynthesis, which is reduced also to 37% of normal. We can also demonstrate that the reduction in M6PR300 is sufficient to increase a lysosomal enzyme secretion.     

Structure-dependent activity of glycyrrhetinic acid derivatives as peroxisome proliferator-activated receptor {gamma} agonists in colon cancer cells

Glycyrrhizin, a pentacyclic triterpene glycoside, is the major phytochemical in licorice. This compound and its hydrolysis product glycyrrhetinic acid have been associated with the multiple therapeutic properties of licorice extracts. We have investigated the effects of 2-cyano substituted analogues of glycyrrhetinic acid on their cytotoxicities and activity as selective peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. Methyl 2-cyano-3,11-dioxo-18beta-olean-1,12-dien-30-oate (beta-CDODA-Me) and methyl 2-cyano-3,11-dioxo-18alpha-olean-1,12-dien-30-oate (alpha-CDODA-Me) were more cytotoxic to colon cancer cells than their des-cyano analogues and introduction of the 2-cyano group into the pentacyclic ring system was necessary for the PPARgamma agonist activity of alpha-CDODA-Me and beta-CDODA-Me isomers. However, in mammalian two-hybrid assays, both compounds differentially induced interactions of PPARgamma with coactivators, suggesting that these isomers, which differ only in the stereochemistry at C18 which affects conformation of the E-ring, are selective receptor modulators. This selectivity in colon cancer cells was shown for the induction of two proapoptotic proteins, namely caveolin-1 and the tumor-suppressor gene Krüppel-like factor-4 (KLF-4). beta-CDODA-Me but not alpha-CDODA-Me induced caveolin-1 in SW480 colon cancer cells, whereas caveolin-1 was induced by both compounds in HT-29 and HCT-15 colon cancer cells. The CDODA-Me isomers induced KLF-4 mRNA levels in HT-29 and SW480 cells but had minimal effects on KLF-4 expression in HCT-15 cells. These induced responses were inhibited by cotreatment with a PPARgamma antagonist. This shows for the first time that PPARgamma agonists derived from glycyrrhetinic acid induced cell-dependent caveolin-1 and KLF-4 expression through receptor-dependent pathways.     

Ligand-directed functional heterogeneity of histamine H1 receptors: novel dual-function ligands selectively activate and block H1-mediated phospholipase C and adenylyl cyclase signaling

The autacoid and neurotransmitter histamine activates the H(1) G protein-coupled receptor (GPCR) to stimulate predominantly phospholipase C (PLC)/inositol phosphate (IP) signaling and, to a lesser extent, adenylyl cyclase (AC)/cAMP signaling in a variety of mammalian cells and tissues, as well as H(1)-transfected clonal cell lines. This study reports that two novel H(1) receptor ligands developed in our laboratory, (-)-trans-1-phenyl-3-dimethylamino-1,2,3,4-tetrahydronaphthalene (trans-PAT) and (+/-)-cis-5-phenyl-7-dimethylamino-5,6,7,8-tetrahydro-9H-benzocycloheptane (cis-PAB), activate H(1) receptors to selectively stimulate AC/cAMP formation and PLC/IP formation, respectively, in Chinese hamster ovary cells transfected with guinea pig H(1) receptor cDNA. trans-PAT and cis-PAB also are shown to be functionally selective antagonists of H(1)-linked PLC/IP and AC/cAMP signaling, respectively. Whereas cis-PAB H(1) receptor activity is shown to be typically competitive, trans-PAT displays a complex interaction with the H(1) receptor that is not competitive regarding antagonism of saturation binding by the standard H(1) antagonist radioligand [(3)H]mepyramine or H(1)/PLC/IP functional activation by histamine. trans-PAT, however, does competitively block H(1)/PLC/IP functional activation by cis-PAB. Molecular determinants for trans-PAT versus cis-PAB differential binding to H(1) receptors, which presumably leads to differential activation of AC/cAMP versus PLC/IP signaling, likely involves stereochemical factors as well as more subtle steric influences. Results suggest the trans-PAT and cis-PAB probes will be useful to study molecular mechanisms of ligand-directed GPCR multifunctional signaling. Moreover, because most untoward cardiovascular-, respiratory-, and gastrointestinal H(1) receptor-mediated effects proceed via the PLC/IP pathway, PAT-type agonists that selectively enhance H(1)-mediated AC/cAMP signaling provide a mechanistic basis for exploiting H(1) receptor activation for drug design purposes.