Forskolin as a Tool for Examining Adenylyl Cyclase Expression, Regulation, and G Protein Signaling

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1 Cellular and Molecular Neurobiology, Vol. 23, No. 3, June 2003 ( C 2003) Review Forskolin as a Tool for Examining Adenylyl Cyclase Expression, Regulation, and G Protein Signaling Paul A. Insel 1,2 and Rennolds S. Ostrom 1 Received October 16, 2002; accepted October 20, 2002 SUMMARY 1. As initially shown by Seamon and Daly, the diterpene forskolin directly activates adenylyl cyclase (AC) and raises cyclic AMP levels in a wide variety of cell types. In this review, we discuss several aspects of forskolin action that are often unappreciated. These include the utility of labeled forskolin as a means to quantitate the number of AC molecules; results of those types of studies, coupled with efforts to increase AC expression, document that such expression stoichiometrically limits cyclic AMP formation by hormones and neurotransmitters. 2. Response to forskolin is also strongly influenced by the activation of AC by the heterotrimeric G-protein, G s.g s -promoted enhancement of AC activity in response to forskolin occurs not only when cells are incubated with exogenously administered agonists that activate G-protein-coupled receptors but also by agonists that can be endogenously released by cells. 3. Such agonists, which include ATP and prostaglandins, serve as autocrine/paracrine regulators of cellular levels of cyclic AMP under basal conditions and also in response to forskolin and to agonists that promote release of such regulators. 4. The ability of forskolin to prominently activate cyclic AMP generation has proved valuable for understanding stoichiometry of the multiple components involved in basal cyclic AMP formation, in enzymologic studies of AC as well as in defining responses to cyclic AMP in cells within and outside the nervous system. KEY WORDS: forskolin; adenylyl cyclase; G-protein-coupled receptors; ATP release; P2Y receptors; stoichiometry; G s. 1 Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California. 2 To whom correspondence should be addressed at Department of Pharmacology, 0636, University of California, San Diego, La Jolla, California ; pinsel@ucsd.edu /03/ /0 C 2003 Plenum Publishing Corporation

2 306 Insel and Ostrom INTRODUCTION As recently reviewed (Beavo and Brunton, 2002), the initial discovery of cyclic AMP (camp) by Sutherland and coworkers set the stage for efforts during the 1960s and 1970s to define the physiologic role of this second messenger in mediating response to hormones and neurotransmitters. Other work emphasized the identification of the molecular components necessary for the stimulation of camp synthesis by the enzyme adenylyl cyclase (AC). In addition to hormonal and neurotransmitter agonists, other entities, such as Mn ++ and NaF (the active form of which was later shown to be fluroaluminate) (Sternweis and Gilman, 1982) were able to stimulate AC activity, in particular in broken-cells and membrane preparations. Certain bacterial toxins, such as cholera toxin and the heat-labile toxin of Escherichia coli, were found to increase AC activity; this action results from the ability of the toxins to catalyze the ADP ribosylation of what was shown to be the heterotrimeric guanine nucleotide (G)-protein, G s, so designated because it promotes stimulation of AC. ADP ribosylation blunts GTPase activity of G s, thereby enhancing its activation by GTP (Cassel and Selinger, 1977). Such observations provided key background information at the time Seamon and Daly identified the diterpene forskolin as a ubiquitous activator of eukaryotic AC (Seamon et al., 1981). Forskolin has proved to be an extremely valuable and widely used reagent: a search on PubMed with forskolin as a key word identifies >15,000 papers. In most cases investigators have employed forskolin as a means to increase camp levels in preparations of membranes, cells, or tissues. Forskolin not only activates AC but also interacts with certain other proteins, including glucose transporters and ion channels; studies with forskolin derivatives demonstrate that such interactions have different structure activity patterns than those observed for activation of AC activity (Morris et al., 1991a; Seamon et al., 1983). The ability of forskolin to interact with AC has been exploited to develop affinity reagents for labeling or purifying the enzyme, sometimes termed C, the catalyst that generates camp (Pfeuffer and Pfeuffer, 1989; Sievert et al., 2002). In this paper, we briefly review some less well-appreciated aspects regarding forskolin as a tool to assess AC and increase camp formation: the utility of radiolabeled forskolin as a means to quantitate the number of molecules of AC and the role of G s and cellularly released agonists in contributing to response to forskolin. We emphasize work from our own laboratory but also note observations by others related to these topics. LABELED FORSKOLIN AS A MEANS TO QUANTITATE AC Shortly after the discovery that forskolin was an efficacious and general activator of camp formation, it was proposed that labeled forskolin might be used to identify AC (Seamon et al., 1984). However, as noted previously, subsequent studies showed that forskolin also interacts with glucose transporters and certain ion channels (Ono et al., 1995; Shanahan et al., 1987; Wadzinski et al., 1987). A number of technical adjustments are required to minimize the latter interactions if one seeks to detect AC (Morris et al., 1991a; Pfeuffer and Pfeuffer, 1989). Of particular note, use of

3 Forskolin s Utility for Studying G s AC Signaling 307 1,9-dideoxyforskolin, a derivative that does not activate AC, is helpful in distinguishing interaction with AC versus binding to other membrane proteins (Laurenza et al., 1992; Morris et al., 1991b). In addition to [ 3 H]forskolin derivatives, iodinated forms, which have higher specific activities, have been used to identify putative AC binding sites by autoradiography with brain preparations (Appel et al., 1992; Robbins et al., 1992). Seamon has reviewed experimental details regarding the use of labeled forskolin to identify AC (Laurenza and Seamon, 1991; Sievert et al., 2002). One generally observes multiple classes of binding sites and even those most readily detectable show relatively low affinity (i.e. in high nm range). The highest affinity sites appear to result not from the binding of forskolin to AC alone but from the interaction of AC with G s. Assays of AC activation in cells, membranes, and purified components have demonstrated that such interaction enhances activation of AC (Bender and Neer, 1983; Daly et al., 1982; Darfler et al., 1982; Dessauer et al., 1997; Green and Clark, 1982a,b). Results from X-ray crystallographic studies directly demonstrate the interaction of G s and C that occurs in the presence of forskolin (Tesmer et al., 1997; Zhang et al., 1997). Thus, when appropriately validated, binding assays with labeled forskolin can be used to detect G s AC complexes. Activation of AC by hormones and neurotransmitters minimally requires three components, receptor, G s, and AC itself (Cerione et al., 1984; May et al., 1985). However, when expressed in native cells, this signaling machinery is much more complexly organized than just those three components. There are multiple varieties of receptors, G s proteins, and ACs, and they are assembled so that activation can occur by various combinations of components. A large number of different G-protein-coupled receptors (GPCRs) activate G s (by promoting exchange of GTP for GDP bound to the α subunit of G s ); well over a dozen such GPCRs can be expressed on a given cell (D. Chalmers, Arena Pharmaceuticals, personal communication, 2002). There are two unique types of the α subunit of G s and a poorly defined number of different G β and G γ subunits that constitute the physiologically relevant forms of the G s heterotrimer. Hydrolysis of G s -bound GTP to GDP can be strikingly enhanced by a GTPase-activating protein, a regulator of G-protein signaling, RGS-PX1, which was recently identified; consistent with that action, RGS-PX1 blunts agonist-promoted stimulation of AC activity (Zheng et al., 2001). Nine different transmembrane isoforms of AC have been identified that can be activated by G s. Forskolin is able to promote activation of each of these, albeit with somewhat less efficacy for AC9 (Hanoune and Defer, 2001). The multicomponent nature of the GPCR G s AC system leads to an important question regarding the ability of hormonal or neurotransmitter agonists to stimulate camp formation: which component is the primary determinant of potency (sometimes, albeit not always correctly, termed affinity, sensitivity, EC 50, K a,ork m ), and efficacy (i.e., maximal response) of such agonists? The answer to this question, which has important physiologic and pharmacologic implications, requires quantitative analysis of each of the components (Ostrom et al., 2000b). Although the kinetics of interaction among components contribute to response, these interactions are in part dependent on the number of each of the components that are expressed in a given target cell or membrane preparation. We have defined the expression of

4 308 Insel and Ostrom Fig. 1. Quantification of the GPCR/G s /AC signaling pathway. Schematic diagram shows the components of the pathway and the number of molecules of each expressed per cell. βar was quantified by radioligand binding, G s by quantitative immunoblot analysis, and AC by [ 3 H]forskolin binding in adult rat cardiac myocytes (Post et al., 1995). Bottom figure shows the relative signal strength based upon the stoichiometry among the components and illustrates that there is not signal amplification along this pathway. Instead, AC expression appears to limit the maximal generation of camp (Gao et al., 1998; Ostrom et al., 2000b). individual components in cells and the impact of various treatments that alter camp generation on such expression (Alousi et al., 1991; Leiber et al., 1993; Jasper et al., 1995; Post et al., 1995). GPCRs can readily be quantified by radioligand binding assays and the α subunit of G s by use of antibodies but it is more difficult to obtain quantitative data regarding expression of AC. Use of labeled forskolin, especially because it is a general activator of multiple AC isoforms, provides a means to identify and quantify high-affinity binding sites, i.e., G s AC complexes (Alousi et al., 1991; Barber, 1988; Post et al., 1995). Results of our studies and those of other workers (MacEwan et al., 1996; Milligan, 1996) have demonstrated that there is a substantial molar excess of G s protein relative to GPCR and that cells express a modestly greater amount of AC compared to G s but a far lower level of AC compared to G s (Fig. 1). Results supporting this idea were obtained with whole (or permeabilized) cells and membrane fractions and indicate that expression of AC plays a key role as a downstream bottleneck for GPCR/G s signaling. While expression of AC appears to be critical for determining the maximal response of G s activation to promote camp formation, GPCR expression plays a key role in determining potency of agonists in the GPCR G s AC pathway (Zhong et al., 1996). Incubation of cells with microtubule inhibitors increases the number of high-affinity binding sites for forskolin, implying

5 Forskolin s Utility for Studying G s AC Signaling 309 enhancement in G s AC coupling by such agents (Leiber et al., 1993). Increasing expression of AC by use of an adenoviral construct increases the number of forskolin binding sites and produces a proportional increase in β-adrenergic agonist-promoted camp formation (Gao et al., 1998). Conversely, extended incubation of cells with agonists decreases the number of agonist-promoted binding sites for forskolin, results consistent with desensitization of the GPCRs (Post et al., 1996). Thus, studies with labeled forskolin provide a useful means to define the number of AC sites and to assess impact of various treatments on G s AC coupling and ability of cells to respond to G s -linked agonists. It will be of interest to determine if labeled forskolin can be used to provide information about the stoichiometry and signaling by isoforms of AC in microdomains, such as caveolin-rich regions of the plasma membrane, which have recently been implicated as sites of GPCR G s AC signaling in a number of cells types (Fagan et al., 2000; Ostrom et al., 2000c,2002; Rybin et al., 2000; Schwencke et al., 1999; Steinberg and Brunton, 2001). FORSKOLIN AS A TOOL FOR CHARACTERIZING G S PROTEIN ACTIVATION OF AC ACTIVITY Another underappreciated use of forskolin is as a means to define basal activation of G s and AC activity. Cells at rest, i.e., under basal conditions, maintain a level of camp that is the consequence of synthesis by AC and degradation by phosphodiesterases. What are the agonists or cellular mechanisms that are responsible for the basal formation of camp? Our data suggest that one class of agonists that determines basal levels of camp are nucleotides released from cells and that act in an autocrine/paracrine manner to activate P2Y receptors and stimulate pathways that ultimately result in increased formation of camp. Various mechanical and chemical perturbations can alter the release of ATP, initiate P2Y receptor signaling, and contribute to basal levels of second messengers (Ostrom et al., 2000a). We first observed this phenomenon when measuring basal and forskolin-stimulated camp production in MDCK cells incubated with or without indomethacin, an inhibitor of cyclooxygenases. Indomethacin, by blocking production of prostaglandins formed in response to cellular ATP release and P2Y receptor activation, lowered the basal activation of G s. Because of the low basal camp levels that one typically observes, the effect of this ATP-prostaglandin pathway on camp production can be difficult to detect. However, in the presence of forskolin, one can dramatically enhance the dynamic range of the response because of the ability of forskolin to potentiate G s - mediated activation of AC (Daly et al., 1982; Darfler et al., 1982; Sutkowski et al., 1994). The result is that even a small amount of G s activation is readily detectable as an increase in camp formation when forskolin is present and, in turn, inhibition by indomethacin is easily observed (Fig. 2). In our studies with MDCK cells, we found that destruction of extracellular ATP with apyrase (an enzyme that rapidly hydrolyzes ATP) inhibits forskolin-stimulated camp production to an extent similar to that observed with indomethacin treatment (Fig. 2). Such results have several important implications for the use of forskolin, especially in studies with intact cells. (1) Response of cells to forskolin can involve

6 Fig. 2. Forskolin is a tool for assessing the basal levels of G s activation. (A) Forskolin-stimulated camp production in MDCK cells is sensitive to cyclooxygenase inhibition (1 µm indomethacin, open bars) and hydrolysis of extracellular ATP (2 U/mL apyrase, hatched bars) but basal levels of camp accumulation are not significantly different than control (paired t test). camp accumulation was measured in the presence of a phosphodiesterase inhibitor using the adenine prelabeling method as previously described (Ostrom et al., 2000a). (B) This schematic diagram illustrates the signaling pathways in MDCK cells that appear to contribute to the basal activation of G s. ATP is released from cells and, activates P2Y receptors that couple to the liberation of arachidonic acid and the production of PGE 2. This prostaglandin then acts in an autocrine/paracrine fashion to activate prostanoid receptors (EPR) coupled to G s. Forskolin response is potentiated by this low level of G s activation, as is evidenced by the effect of indomethacin or apyrase (panel A). Not shown in this simplified schematic are P2Y 11 receptors that are also activated by released ATP and can couple directly to the activation of G s (Torres et al., 2002).

7 Forskolin s Utility for Studying G s AC Signaling 311 activation of GPCRs, such as nucleotide and prostaglandin receptors that link to G s activation. Thus, cellular generation of camp in response to forskolin can include a contribution by GPCR activation even though one has not intentionally added a GPCR agonist. Although our studies have emphasized release of ATP, work by others indicates that UTP can also readily be released from cells (Lazarowski et al., 1997; Lazarowski and Harden, 1999); UTP is equipotent with ATP in activating P2Y2 receptors and increasing camp in MDCK cells, and perhaps others. (2) Activation of G s by GPCRs contributes to forskolin-stimulated camp generation in cells because of G s -forskolin potentiation of AC activity. Even though evidence for this idea has been obtained in multiple experimental systems, many investigators who observe increases in camp in response to forskolin mistakenly assume such increases imply an activation of AC independent of upstream components. The structural basis of this G s -forskolin potentiation is evident from crystallographic studies of AC. The functional unit of AC is formed by two domains (termed C1 and C2) arranged in a semisymmetrical mirror image of each other (Dessauer et al., 1998; Tesmer et al., 1997). The C1 domain contains the catalytic and G i binding sites while the C2 domain provides the G s binding site as well as a pseudocatalytic site at which forskolin binds. Thus, it is not surprising that G s interaction with AC favors both substrate catalysis at the active site and binding of forskolin at the pseudocatalytic site (Dessauer et al., 1998). It is interesting to note that the ability of indomethacin or apyrase to substantially blunt camp generation in response to forskolin may relate to the observation, originally noted by Daly nearly 20 years ago, that forskolin stimulates less camp production in broken-cell AC activity assays as compared to intact cell assays (Daly, 1984). When viewed in the context of our recent results, such findings may relate to the loss (in broken-cell preparations) of a contribution of agonists formed and released by intact cells and that act via G s to enhance the stimulation of AC by forskolin. In addition, the ability of forskolin to enhance G s -mediated camp formation also provides a means to elicit camp stimulatory responses of weak or partial agonists; this response to forskolin demonstrates yet another potential use of the diterpene to help define the action of drugs that act on the G s AC system (Jasper et al., 1988). CONCLUSIONS The results that we have discussed herein provide examples of the utility of forskolin as a compound for the study of AC and camp formation, especially in experiments with intact-cell preparations. The diterpene can be used to quantitate the number of AC molecules and to assess the impact of various treatments on AC expression. Studies of forskolin-stimulated camp formation in intact cells reveal a contribution by endogenously formed agonists that act directly or indirectly via GPCRs and a key role for G s stimulation in what is measured as forskolin response. Overall, our findings add to the considerable literature on forskolin, which was initiated by the seminal early studies by Daly, Seamon, and coworkers. Even though forskolin has been used for over 20 years, it remains and will continue to be an important agent in studies of the molecular basis of drug action.

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