By W. Sanford. Ryokan College.

In the second lithium comparison study (88) 70% and colleagues study had been hospitalized sinequan 25 mg sale. Second purchase cheap sinequan on line, the of 52 hospitalized patients randomized to lithium or CBZ definition of relapse differed between the studies purchase sinequan with mastercard. In the dropped out of the trial by 8 weeks owing to lack of efficacy buy sinequan with visa. Two studies an initial year of treatment with lithium or CBZ, a crossover compared CBZ with chlorpromazine in the treatment of to the alternate drug in the second year, followed by a third acute mania (34,71). In the first comparison trial, 60 acutely year on the combination (23). Among evaluable patients, manic patients were randomized to either agent in a 6-week 13(31%) of 42 lithium-treated patients relapsed within 1 trial (71). There were no significant differences in efficacy year compared with 13 (37%) of 35 CBZ-treated patients. As in the previous study, a higher percentage of 28 patients receiving chlorpromazine. In the second study of patients receiving CBZ withdrew because of adverse (34,37) patients were randomized to CBZ (n 15) or events. The percentage of patients who had moderate or chlorpromazine (n 19) in a 3-week trial. Response was marked improvement on the CGI was not significantly dif- assessed in 26 patients who completed the trial. Patients ferent: 33% on lithium, 31% on CBZ, 55% on the combi- treated with CBZ (n 15) or chlorpromazine (n 11) nation; however, on a variety of measures of mania, lithium had comparable improvement. Finally, patients with a history were rated as displaying at least moderate improvement and of rapid cycling responded significantly better to the combi- 59% for the chlorpromazine group. In summary, data from nation (56% response) compared with lithium (28%) or these five trials suggest that CBZ is superior to placebo and CBZ (19%). STANDARD ANTIPSYCHOTICS Maintenance Acute Mania The efficacy of CBZ in the prevention of recurrent affective episodes has been a matter of controversy (22,77). This There is one double-blind, placebo-controlled study of a controversy rests in part on the heterogeneity among the standard antipsychotic in the treatment of acute bipolar early controlled maintenance studies (22,54,74,101), and mania (50). In that study, 13patients were randomized to the availability of only one placebo-controlled maintenance chlorpromazine (1,200 mg per day), imipramine (300 mg trial (70). Interpretation of this latter study is also limited per day), or placebo for 7 weeks. Response was assessed by the use of adjunctive rescue medications other than lith- using a global scale ranging from 9to 9. Chlorproma- ium and CBZ to treat breakthrough symptoms. The liberal zine was significantly superior to placebo and imipramine use of these adjunctive treatments thus limits the degree to on global outcome (6. Two recent large prospective, double-blind, long-term maintenance studies provide new data comparing the effi- Maintenance cacy of CBZ with lithium (23,33). In the first study (33), 144 patients were randomized to lithium (n 74; mean There are no parallel group, double-blind randomized SD serum level, 0. Affective relapse, hospitalization, need for supplemen- tal medication, and adverse events requiring treatment dis- continuation were used to define treatment failure. Using ATYPICAL ANTIPSYCHOTICS survival analysis, there were no statistically significant differ- ences between the two treatment groups in time to episode Clozapine recurrence or hospitalization. However, significantly more Acute Mania patients receiving CBZ required supplemental medications for symptomatic recurrences and experienced adverse events No double-blind, randomized controlled trials of clozapine requiring treatment discontinuation. In a secondary analysis in the treatment of acute bipolar mania have been pub- of predictors or response, patients with classical features (bi- lished. Clozapine has been reported to be an effective anti- polar I patients without mood-incongruent delusions and manic agent in three open-label, prospective studies (6,14, comorbidity) had a lower rehospitalization rate with lithium 18). The first two studies evaluated treatment-refractory bi- than with CBZ (32). For patients with nonclassical features polar patients. In the first (6), 13(87%) of 15 acutely manic (mixed states, bipolar II, and NOS) a trend in favor of CBZ patients who had failed to respond to a minimum 6-week was found. In the second study (14), 25 acutely manic pa- 49% compared with placebo at 24%. However, olanzapine tients with bipolar or schizoaffective disorder, all of whom did not separate from placebo on the YMRS until the third had failed to respond to or tolerate lithium, VPA, or CBZ, week of treatment. Eighteen (72%) patients dis- (n 60) for up to 4 weeks (98). This study used a higher played marked improvement on the YMRS and eight (32%) initial starting dose of olanzapine, 15 mg per day, and con- on the BPRS, defined as more than 50% reduction in total comitant lorazepam use was further restricted to 2 mg per score on either scale. Bipolar patients compared with schizo- day for the first 10 days. The olanzapine group again dis- affective patients, and non-rapid cyclers compared with played significant improvement on the YMRS, CGI-BP se- rapid cyclers, exhibited significantly greater improvement verity of mania, PANSS total and positive symptom scores on the BPRS. In the third study (18), 30 hospitalized acutely compared with the placebo group. These differences were manic patients were randomized to clozapine (mean 166 evident by week one (the time of the first rating) and sus- mg per day) or chlorpromazine (mean 310 mg per day) in a tained throughout the trial. Although clozapine-treated patients displayed exhibited significantly higher response (65% versus 43%, significantly lower YMRS scores after 2 weeks, there were respectively) and remission (61% versus 36%, respectively) no significant differences between the two groups at the rates than placebo-treated patients. There are no randomized double-blind controlled studies In the third controlled trial, 30 inpatients with acute of clozapine in the maintenance treatment of BD. Suppes mania were randomized to olanzapine (n 15) or lithium and co-workers randomized 85 patients with DSM-IV crite- (n 15) for 4 weeks (7). Olanzapine was administered as ria for schizoaffective or BD with treatment-refractory ill- a fixed dose of 10 mg per day and lithium at 400 mg BID ness to add-on clozapine or treatment as usual (94). Concomitant lorazepam 1 year, patients receiving clozapine displayed significant re- 4 to 12 mg per day was permitted throughout the 4-week ductions in measures of mania, psychosis, and global im- trial. There were no significant differences between the two provement compared with patients receiving treatment as treatment groups on any of the primary outcome mea- usual. In addition, total medication use decreased signifi- sures—the Mania Scale and BPRS total scores and the CGI cantly in the clozapine group. However, olanzapine-treated patients trial confirmed earlier reports suggesting that clozapine ex- displayed significantly greater improvement than lithium- erted long-term mood-stabilizing effects in patients with treated patients on the CGI severity scale at the end of 4 treatment-refractory BD (102). Olanzapine Maintenance Acute Mania There are no controlled trials of olanzapine in the mainte- nance treatment of BD published to date. In an open-label The efficacy of olanzapine in the treatment of acute bipolar 52-week extension trial following the initial placebo-con- mania has been established in three double-blind, controlled trolled acute mania study, none of the 98 patients who trials (7,98,99). In the first of two placebo-controlled stud- participated developed tardive dyskinesia during long-term ies, 139 inpatients with bipolar I disorder were randomized treatment (99). Olanzapine was begun at 10 mg per day and adjusted by 5-mg per day increments within a range of 5 Risperidone to 20 mg per day; the median modal dose was 15 mg per Acute Mania day. Concomitant lorazepam up to 4 mg per day was per- mitted for the first 7 days as needed for agitation; 2 mg per There are two double-blind randomized active comparator day was permitted for the subsequent 3days. The olanzap- studies of risperidone in the treatment of acute bipolar ine group displayed significant improvement on the YMRS, mania (84,86). In the first study, 45 inpatients were ran- CGI-BP severity of mania, and the PANSS total and posi- domized to risperidone 6 mg per day (n 15), haloperidol tive symptom scores compared with the placebo group. Patients receiving placebo exhibited significantly for up to 4 weeks (86). There were no significant differences greater improvement in YMRS total scores compared with among the three treatment groups in reductions on the patients receiving gabapentin. YMRS, BPRS, CGI, and GAF from baseline to endpoint In the second controlled trial, 28 patients with bipolar (LOCF). In the second trial, 158 inpatients receiving lith- I(n 13) or bipolar II (n 15) disorder received 6-week ium or VPA were randomized to adjunctive therapy with crossover trials of gabapentin, lamotrigine, or placebo (29) risperidone 1 to 6 mg per day (n 52), haloperidol (n The reduction in manic symptoms as measured by the CGI- 53), or placebo (n 51) for up to 3weeks (84). Patients BP was not significantly different among the three treat- receiving risperidone or haloperidol displayed significantly ments. However, manic symptoms were quite low at base- greater improvement at 1 week, 2 weeks, and at endpoint line, raising the possibility that meaningful differences (LOCF), but not at 3weeks of treatment on the YMRS among the three groups might not have been detected.

Extensions of these experi- preoptic area order 25mg sinequan, and substantia innominata is significantly ments to mice lacking other subunits of the nicotinic recep- lower than in the septum and nucleus basalis cheap sinequan 25mg on-line. This observa- tor should allow identification of the receptor subtypes that tion has led to the hypothesis that activation of primarily are activated by smoking in humans and result in tobacco noncholinergic neurons is responsible for producing sleep addiction sinequan 75 mg low cost. An interesting effect of ACh on neuronal survival after basal forebrain stimulation (33) purchase sinequan 25mg with amex. These noncholinergic was demonstrated in mice lacking the 2 nAChR subunit neurons are believed to be GABAergic and achieve their (32). Mice that lack this cholinergic-receptor subtype show effects through inhibition of cholinergic basal forebrain progressive neuronal loss with age in cortical and hippocam- neurons and neurons within the brainstem reticular forma- pal brain areas, which appears to lead to age-related impair- tion. In contrast, stimulation of the nucleus basalis or septal ments in spatial learning. These experiments demonstrate nucleus produces behavioral activation and cortical ACh that the effects of ACh on cognition, antinociception, loco- release, and this is consistent with the notion that basal motion, and overall neuronal activity are differentially me- forebrain cholinergic neurons are involved in behavioral diated through the various subtypes of muscarinic and nico- arousal (activation), whereas noncholinergic basal forebrain tinic receptors, and that the various roles of ACh may be neurons are involved in regulating the sleep state. These separated pharmacologically, suggesting new targets for ra- two effects are related (sleep vs. ROLE FOR CHOLINERGIC NEURONS IN AROUSAL AND SLEEP ROLE FOR CHOLINERGIC NEURONS IN MOTIVATION AND REWARD Traditionally, the basal forebrain complex, the primary source of cholinergic innervation to the telencephalon (Fig. Cholinergic neurons have also been implicated in motiva- 1. Either lesions or electric stimulation of subregions of that nAChRs are involved in motivation and reward is that the basal forebrain can facilitate sleep and synchronize the nicotine is abused by humans and is reinforcing in animals EEG, and cholinergic drugs regulate EEG synchrony (33). The effects of nicotine on tests Moreover, a correlation between cortical ACh release and of reinforcement and behavioral sensitization are primarily the state of behavioral activation or sleep has been observed mediated through the mesolimbic dopamine system (39). Thus, it was hypothesized that cholinergic input Indeed, the ventral tegmental area (VTA) may be sufficient to the neocortex from the basal forebrain is critical for regu- to mediate the reinforcing properties of nicotine, as local lating arousal (see ref. These neurons largely do not inner- preference (41). Chapter 1: Acetylcholine 7 Basal forebrain cholinergic neurons may also be involved lesions increased sucrose consumption, similar lesions did in modulating cortical processing of stimuli with condi- not affect discrimination or contrast effects (57). Neverthe- tioned or unconditioned rewarding properties because these less, the hypothesis of Winn (58) is that lesions of the PPT neurons are more responsive to stimuli with a high incentive affect responding for rewarding stimuli similarly to lesions value. Novel stimuli that typically elicit orienting responses of the frontal cortex, so that the role of the PPT, like that of and attention in animals increase cortical ACh release, but the basal forebrain, is expanded into higher-order cognitive this effect is diminished with repeated exposure if the stimu- processes. In contrast, if the stimulus is repeatedly paired with an incentive stimulus (e. Pontomesencephalic cholinergic neurons are also The hypothesis of cholinergic involvement in learning and involved in motivation and reward, although these effects memory processes arose from several findings. Both destruc- are likely mediated, in part, by projections to the dopamine tion of the basal forebrain complex and the administration neurons within the VTA (44,45). While a significant proportion of the The original finding that lesions of the basal forebrain PPT neurons that project to the tegmental dopamine neu- could produce deficits in a variety of cognitive tasks sug- rons are noncholinergic (44), the cholinergic input per se gested a role for ACh in cognitive function. Electrolytic, appears to stimulate dopamine neurons (47). Thus, ascend- radiofrequency, or nonspecific excitotoxic lesions of cholin- ing projections from the PPT to the dopamine cells may ergic subnuclei within the basal forebrain (particularly the regulate the ability of mesostriatal dopamine neurons to medial septum/diagonal band) profoundly impair perfor- affect incentive/motivational processes. These deficits appeared modulate the rewarding qualities of addictive drugs. Lesions to be reversed following regeneration of cholinergic projec- of the PPT reduce the self-administration of nicotine (48) tions across a bridging graft (61) or after grafting of ACh- and opiates (49). Moreover, conditioned place preference producing cells in the hippocampus (62). These findings for food, opiates (50), morphine (51), and amphetamine have been interpreted as support for the hypothesis of cho- (52) is blocked or reduced by PPT lesions, whereas cocaine- linergic involvement in cognitive functions; however (as induced reward is unaffected (53). Although the mesolimbic with arousal and sleep), noncholinergic neurons within the dopamine pathway is known to be involved in drug reward basal forebrain may likewise be involved in these effects, (see ref. It is also not known whether the effect Novel approaches for selectively destroying cholinergic of PPT lesions on these processes is mediated through pro- neurons depend on the differential sensitivity of basal fore- jections to areas other than the dopamine cell groups within brain neurons to excitotoxins and new types of immunotox- the VTA. Systematic studies have demonstrated that cholinergic The PPT may have another, more critical, role in motiva- and noncholinergic neurons within the basal forebrain are tion and reward via afferent inputs from the striatum (55). Based on the results of these stud- ulant-induced orofacial stereotypy, yet no difference is ob- ies, new methods for preferentially destroying cholinergic served in stimulant-induced locomotion or other measures neurons have been described (63). These data may implicate the porin toxin has been developed that takes advantage of the PPT (and its innervation from the striatum) in response fact that basal forebrain cholinergic neurons are particularly selection when discrimination is involved because the dis- enriched with low-affinity receptors for nerve growth factor ruption of responding for conditioned reinforcement re- (64). The toxin selectively binds to the receptor for nerve sulted from decreased discrimination of response between growth factor and then kills the neuron expressing the recep- a lever associated with reinforcement and an inactive lever tor. More excitingly, recent studies suggest that IgG–sa- (56). However, a recent study found that although PPT porin can be used to destroy the cholinergic innervation of 8 Neuropsychopharmacology: The Fifth Generation of Progress conditioning but impairments in discrete cue (trace) condi- tioning (69). Both sets of data may suggest that the atten- tional processing of discrete stimuli is disrupted following cholinergic depletion from posterior cortical regions. It is possible, however, that the depletion of ACh from caudal or rostral cortical regions alone may be insufficient to impair performance of some tasks, whereas combined depletions may have more than additive effects (70). Other investigators have further argued that the choliner- gic innervation of rostral (e. Direct pharmacologic manipulation of basal forebrain neurons has been used to alter activated cholinergic efflux in the frontal cortex and performance of tasks related to stimulus process- ing or detection (72). Selective excitotoxic lesions or phar- macologic manipulation of the nucleus basalis has also been reported to impair performance in a five-choice serial reac- tion task that requires animals to detect and respond to brief visual stimuli (73). Interestingly, the observation that appetitive pavlovian learning for a discrete cue is enhanced after nucleus basalis lesions (74) suggests that attentional processing of discrete cues may not be affected by depletion of ACh from the rostral neocortex except when divided attention is required. The findings of these latter studies are also bolstered by advances in the measurement of ACh FIGURE 1. Acetylcholinesterase staining of the nucleus basalis magnocellularis after infusion of saline solution or AMPA to de- transmission in vivo, which allows investigators to quantify stroy cholinergic neurons preferentially. Low concentrations of directly the extent of the lesions produced by the toxins for the glutamatergic agonist AMPA selectively destroy cholinergic the first time (75). Taken together, the available data seem neurons (measured by acetylcholinesterase staining) and spare -aminobutyric acid (GABA) neurons (left). In contrast, control to suggest that basal forebrain cholinergic neurons are capa- sections show robust acetylcholinesterase staining after infusion ble of regulating the cortical processing of sensory stimuli of saline solution (right). This process allows more specific cholin- within a variety of domains, which may be explained by a ergic lesions to be generated, so that the function of the neurons role for basal forebrain ACh in the regulation of cortical in behavioral processes can be clarified. Tegmental cholinergic neurons have also been implicated in cognitive processes (58,76). Although some of the effects terminal regions into which the toxin is injected (65). These of PPT lesions on learning and memory may be related to methods have been applied to studies of learning and mem- generalized anxiety (76), PPT lesions also produce a set of ory in an attempt to qualify earlier findings. Essentially, selective damage to cholin- memory performance does not seem to be affected by de- ergic neurons of the basal forebrain has failed to produce struction of the PPT (77). The position of the PPT as a the retrograde or anterograde amnesia or deficits in learning modulator of dopaminergic systems (which affect frontal that have been reported to result from nonspecific lesions cortex function), in addition to the influence of the frontal of the basal forebrain (59,66). Previously, the medial septal/ cortex on the PPT (mediated through the striatum), sug- diagonal band nuclei and their projections to posterior corti- gests that this nucleus is in an excellent position to affect cal regions were thought to be critical for spatial learning the functions of the frontostriatal system. By means of saporin le- that attempts to control for the extent and selectivity of sions, however, cholinergic depletion within the hippocam- PPT lesions is necessary. Moreover, selective excitotoxic lesions of the medial Although lesions of cholinergic nuclei have implicated ACh septum/diagonal band produce enhancements in contextual in various behavioral processes, it is also of interest to deter- Chapter 1: Acetylcholine 9 mine which cholinergic-receptor subtypes mediate these re- produced dose-dependent performance impairments when sponses to ACh. Systemic infusions of the muscarinic-recep- administered 45 minutes before testing on the delayed alter- tor antagonists atropine and scopolamine produce an nation task, suggesting that decrements in cholinergic stim- amnesic syndrome in humans (78), monkeys (79), and rats ulation of muscarinic receptors result in cognitive dysfunc- (80). Several lines of evidence suggest that multiple central tion. FG7142 (20 mg/kg) significantly elevated prefrontal nervous system structures, including the medial septum/ cortical ACh release in vivo (measured in parallel studies), diagonal band region, are critical in mediating the effects and FG7142 on its own impaired delayed alternation per- of muscarinic drugs on mnemonic functions (80). Interestingly, the fact that coadministration of of muscarinic-receptor antagonists into a variety of cortical FG7142 and scopolamine did not affect the slope of the regions, including the hippocampus, prefrontal cortex, and dose–response curve for scopolamine suggests that these amygdala, can impair the cognitive functions associated two drugs act on different mechanisms to impair delayed with these respective regions (81).

GPCRs can be detected in situ in cell or tissue preparations using immunochemical techniques and receptor-specific an- Biochemical Methods to AssaySpecific tibodies sinequan 25mg with mastercard. Antibodies that recognize the native receptor pro- Receptor Trafficking Processes tein can be used to examine the localization of endogenously expressed receptors purchase sinequan overnight, whereas epitope-tagging methods (see Whereas microscopic imaging can readily provide a great above) can be used to detect mutated versions of the receptor deal of qualitative information about GPCR localization protein or as a means to detect recombinant receptors for and trafficking buy sinequan us, it can be quite challenging to quantitiate A B FIGURE 22 buy sinequan 75mg free shipping. Visualization of HA epitope-tagged dopamine D1 receptors in transfected cells, using a fluorochrome-labeled secondary antibody and fluorescence microscopy. The ability of this receptortoundergoregulatedinternalizationis indicatedbythedopamine-inducedredistribution of immunoreactive receptors from the plasma membrane (visualized as linear staining at the cell periphery) to endocytic vesicles (visualized as punctate structures located throughout the cytoplasm). In addition to being extremely a specific subcellular localization or to measure accurately useful for examining posttranslational modifications of the rate or extent of specific trafficking processes. The im- GPCRs, in some cases it is possible to use these techniques portance of these processes has motivated the development to isolate receptor-containing complexes that presumably of biochemical methods for examining GPCR trafficking. The In addition to their utility for receptor localization, antibod- basic idea is to immunopurify a specific GPCR from cell ies specifically recognizing GPCRs facilitate biochemical or tissue extracts (or from a partially purified subcellular studies of GPCR trafficking using techniques adapted from fraction prepared from a cell or tissue lysate) using an anti- other areas of cell and molecular biology. For example, one body recognizing the native receptor or an engineered epi- method that has been extremely useful for quantitative stud- tope tag, and then to analyze proteins specifically associated ies of GPCR endocytosis is cell-surface biotinylation cou- with this complex using a different antibody. In general, pled with immunoprecipitation of receptors. Proteins pres- this is accomplished by immunoprecipitation of the receptor ent in the plasma membrane of cells can be specifically followed by analysis of associated proteins in the complex labeled by incubating intact cells in the presence of biotin by immunoblotting with the appropriate additional anti- coupled to an activated ester, which is membrane-imper- body. In some cases, the protein complexes are sufficiently meant and therefore forms a covalent bond only with ex- stable that they remain associated through the initial immu- posed amine moieties present in plasma membrane proteins. In other cases this is not true, In general, biotinylation in this manner does not adversely and the complexes dissociate before the receptor can be affect GPCR function, allowing biotinylation to be used as purified from the extract. In this case, various chemical a chemical tag for surface receptors. Using variations of this basic biochemistry, it is possi- teraction with heterotrimeric G proteins (52) and with - ble to measure a wide variety of membrane trafficking pro- arrestins (53), and to examine the regulation of these protein cesses. For example, internalization of GPCRs has been interactions by ligand-induced activation of the receptor. Recent studies provide strong support for this idea and, specifically, provide evidence for homo- and heterodimeri- Methods for Examining Specific Protein zation of individual GPCRs in vivo. This principle is per- Interactions Involved in GPCR Function haps best established for receptor tyrosine kinases, where it and Regulation is well established that oligomerization of receptors is re- A salient lesson emerging from recent cell biological studies quired for appropriate ligand-dependent signal transduction is that GPCR signal transduction can be viewed, in essence, (54). A relatively early hint that GPCRs may also undergo as a dynamically regulated network of protein–protein in- oligomerization came from studies of the 2-adrenergic re- teractions that occur in specific subcellular locations. There- ceptor using epitope-tagging techniques, where it was ob- fore, an important goal of current and future research is to served that receptors tagged with one epitope could specifi- define these critical protein interactions and elucidate their cally coimmunoprecipitate receptors tagged with a distinct temporal and spatial regulation in intact cells and tissues. More recently, evidence for oligomerization of many Coimmunoprecipitation Techniques to GPCRs has been reported. A particularly compelling exam- Examine Defined Protein Interactions ple of this is the recent observation that distinct subtypes with GPCRs in Intact Cells of GABA-B receptor hetero-oligomerize in cells, and that As discussed above, it is possible to rapidly purify GPCRs oligomerization is essential for the formation of recombi- from cell or tissue extracts using receptor-specific antibodies nant receptors possessing the functional properties charac- 22: G-Protein–Coupled Receptors 285 teristic of native GABA-B receptors observed in vivo interactions. A cDNA library prepared from a tissue of inter- (57,58). Both the bait and prey expressed opioid receptors (59). In a recently published study transcription of the reporter gene. However, if the fused (60), glutathione S-transferase (GST)-fusion proteins en- bait and prey polypeptides form a sufficiently stable pro- coding the C-terminal tail of the D5 receptor were shown tein–protein interaction, they bring their corresponding to interact with the GABA-A receptor present in rat hippo- DNA binding and transcriptional activation domains into campal extracts. Additionally, using an antibody recogniz- close proximity, thus reconstituting transcriptional activa- ing the dopamine D5 receptor, it was possible to coimmu- tion of the reporter gene. Transformed yeast cells containing noprecipitate the GABA-A receptor from cell extracts. In addition to known proteins that mediate and regulate Protein interactions suggested to occur by the yeast two- GPCR signaling (heterotrimeric G proteins, GRKs, ar- hybrid system can be examined using various in vitro bio- restins), which were originally identified by functional as- chemical techniques, such as affinity chromatography facili- says using biochemical purification, cDNA cloning meth- tated by GST-fusion proteins. In addition to serving as an ods have facilitated the identification of additional protein independent assay for previously defined candidate interact- interactions with GPCRs that were completely unantici- ing proteins, this method can be used to identify novel pated (61). These novel protein interactions, while their protein interactions with GPCRs de novo (63). In this functional relevance remains unclear in many cases, are of method a DNA encoding a polypeptide sequence of interest great interest and potential therapeutic importance as drug is fused to GST using standard cDNA cloning techniques targets. The GST Of the many techniques for identifying novel pro- portion of the fusion protein allows the efficient immobili- tein–protein interactions developed over the last 10 years, zation of the protein by binding to agarose beads covalently interaction cloning methods such as the yeast two-hybrid derivatized with glutathione. Proteins from a cell or tissue system (62) have been particularly useful for studies of extract that bind to the fusion protein then can be isolated GPCRs. In the yeast two-hybrid system, protein interac- as an immobilized protein complex by affinity chromatogra- tions are detected by their ability to reconstitute the activity phy. A transcrip- shown recently (64) that the third cytoplasmic loop of the tion factor such as GAL4 can be divided into two domains: dopamine D2 receptor binds specifically to spinophilin, a a DNA binding domain and a transcriptional activation large cytoskeleton-associated protein that also binds to pro- domain. For the transcription factor to be active, these two tein phosphatase-1. A polypeptide sequence for which one phy using GST-fusion proteins. However, even in the event that extensive colocalization is observed, immunocytochem- ical techniques of this sort do not provide direct evidence for a physical interaction between candidate proteins. Coimmunoprecipitation techniques, as discussed above, A provide a useful method for addressing this question. How- ever, demonstrating that a specific protein association can occur in vivo is only the first step in the process of assessing the potential physiologic relevance of a novel protein inter- action, as this method generally does not provide any infor- mation regarding the possible functional activity of a candi- date protein interaction. Addressing this question can be a challenging task that involves creative application of diverse techniques and functional assays. Examples of novel protein interactions with GPCRs for which compelling functional data exist include the aforementioned interaction of the D2 dopamine receptor with ABP280 (65) and interaction of the 2-adrenergic receptor with NHERF/EBP50-family B proteins (51,63). Schematic diagram of G-protein–coupled receptor (GPCR) signaling. Following agonist binding, GPCRs activate heterotrimeric G proteins (G), which then regulate the activity of specific cellular effectors. Followingagonist binding,GPCRs canassoci- ate with members of diverse families of intracellular proteins, Unexpected Signaling, Cross-Talk, and including heterotrimeric G proteins (G), polyproline-binding pro- Transactivation Involving GPCRs teins such as those containing SH3 domains (SH3), arrestins (Arr), G-protein–coupled receptor kinases (GRK), small guanosine tri- (Fig. These Another line of evidence suggesting the existence of func- interactions allow GPCRs to initiate multiple intracellular signal- tionally relevant, novel protein interactions involving ing pathways, with each subtype of receptor likely coupled to GPCRs comes from recent work by several labs suggesting a relatively unique set of effectors. Heptahelical receptor signaling: beyond the G pro- that unanticipated functional interactions can occur be- tein paradigm. The RTK family includes the epidermal growth factor receptor (EGFR), the first receptor shown to have intrinsic tyrosine mechanism of cross-talk involves the formation of hetero- kinase activity (67,68). For tide growth factors (such as EGF) to the extracellular do- example, recent studies suggest that the nonreceptor tyro- main of the RTK, it has been observed recently that certain sine kinase c-Src can associate with the 2-adrenergic recep- GPCRs can initiate signaling cascades traditionally thought tor and the -arrestin in endocytic membranes, thus me- to be controlled by RTKs. For example, several GPCRs can by c-Src-mediated phosphorylation of co-endocytosed mediate transactivation of coexpressed EGFRs, thus stimu- EGFR (72). One Visualization of Protein Localization and mechanism of GPCR-mediated transactivation involves the Interaction in Living Cells activation of a membrane-associated metalloproteinase, which cleaves the EGF precursor protein to generate in- As discussed above, immunochemical methods are useful creased amounts of ligand for the EGFR (70). Another for examining the localization of proteins in intact cells. Indeed, we view newer molecular and because they require disruption of the cell membrane and cell biological approaches as complementing, rather than prolonged incubation of specimens with antibodies used to replacing, the sophisticated pharmacologic methods that detect the receptor of interest. The discovery of proteins have been developed over the years since the discovery of from certain marine animals that have high levels of intrinsic receptors as important drug targets. These proteins, such as the green of cDNAs encoding many G-protein–coupled receptors. This is accomplished by made it practical to produce large amounts of receptor pro- using site-directed mutagenesis to create a fusion between tein for pharmacologic, biochemical and biophysical studies. The localization of the fusion protein can be cise atomic determinants of receptor-ligand interaction and examined in intact cells using fluorescence microscopy. Ex- for understanding protein conformational changes involved amples of this methodology include the visualization of li- in receptor activation and regulation. Continued progress gand-induced endocytosis of a GFP-tagged 2-adrenergic in this important area may lead to entirely new concepts and receptor in living cells and visualizing the dynamic recruit- methods relevant to therapeutic drug design. Site-directed ment of GFP-tagged -arrestin from the cytoplasm to the mutagenesis techniques complement structural and bio- plasma membrane induced by activation of various GPCRs physical approaches and have enabled, in the absence of (75,76).

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