Gi-Mediated Stimulation of Type II Adenylyl Cyclase Is Augmented by G,-Coupled Receptor Activation and Phorbol Ester Treatment

Home , Adenylyl cyclase, Bombesin receptor

The Journal of Neuroscience, February 15, 1996, 76(4):1317-l 323

Rachel C. Tsu and Yung H. Wong Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

Synergism between G,- and Gi- or G,-dependent signaling Activation of protein kinase C (PKC) thus appears to relieve the pathways has been demonstrated in the stimulation of type II requirement for the presence of activated 01,. Stimulation of adenylyl cyclase (AC-II). Provision of activated 01, is known to PKC via G,-coupled receptors also allowed G,-coupled recep- allow numerous Gi-coupled receptors to stimulate AC-II and to tors to activate AC-II. Coexpression of the ml muscarinic potentiate the responses to G,-coupled receptors. To explore receptor with the dopamine-D, receptor permitted dopamine to possible interactions between Gi- and G,-coupled receptors stimulate AC-II in the presence of carbachol. The phorbol ester- that are independent of (Y*, the activity of AC-II was determined permissive and as-independent stimulation was mediated by after the activation of Gi- and G,-regulated pathways. Human G-protein Pr subunits because it was blocked by the Pr scav- embryonic kidney 293 cells were transiently cotransfected with engers O(~ and /3-adrenergic receptor kinase. These results cDNAs encoding AC-II and various G-protein-coupled recep- show that AC-II can efficiently integrate signals generated by tors. Agonist-bound G,-coupled receptors (including the formyl G,- and G,-coupled receptors via a mechanism that is inde- peptide, dopamine-D,, and S-opioid receptors) stimulated pendent of G,. AC-II activity in the absence of activated a,, provided that the Key words: adenylyl cyclase; CAMP; G-proteins; phorbol es- cells were treated with 100 nM phorbol 12-myristate 13-acetate. ter: protein kinase C; signal transduction

Signal processing is a complex and vital function of the nervous AC-II can also be activated by external signals that are routed system. Many external signals in the form of neurotransmitters through protein kinase C (PKC). Both phorbol esters (Jacobowitz and hormones have to be integrated and processed by the neu- and Iyengar, 1994) and agonists that act on G,-coupled receptors rons. An example of how intracellular macromolecules convert (Lustig et al., 1993) are known to stimulate AC-II. These stimu- extracellular signals into a unified message is given by the type II latory effects are attributed to the activation of PKC. It has been isoform of adenylyl cyclase (AC-II), which is abundant in the brain shown that type II as well as type VII adenylyl cyclases are (Feinstein et al., 1991). AC-II is known to process coincident activated after phosphorylation by PKC (Jacobowitz et al., 1993; signals from receptors that are coupled to G,-, G,-, or G,-proteins Watson et al., 1995). The phorbol ester-induced stimulation of (Lustig et al., 1993). Like the other adenylyl cyclase isoforms, the AC-II is distinct from that seen with o(, and Pr subunits, and type II enzyme can be directly stimulated by the GTP-bound cy synergistic effects arising from the two pathways have been re- subunit of G, (Tang and Gilman, 1992). Activation of AC-II via ported (Lustig et al., 1993). Although acting via different mecha- the Gi pathway is more complex. In the current mechanistic nisms, signals generated from G,- and G,-coupled receptors nev- model, activation of a G,-coupled receptor leads to the release of ertheless can converge on the AC-II. In the present investigation, Pr subunits from Gi, and the freed Pr subunits can then stimulate we asked whether AC-II can integrate coincident signals from Gi- AC-II. However, it was found that the presence of activated G,, and G,-coupled receptors. Our results indicate that receptors is absolutely required for the /?y subunits to stimulate AC-II coupled to Gi- and G,-proteins can stimulate AC-II in a syner- (Federman et al., 1992). Thus, when both G,- and G,-regulated gistic manner, thereby providing another level of complexity in the pathways are simultaneously stimulated, the activity of AC-II is regulation of intracellular CAMP. enhanced. Among the various G,-coupled receptors, the dopamine-D,, adenosine-A,, p- and S-opioid, melatonin-lc, com- MATERIALS AND METHODS plement factor C5a, and formyl peptide receptors are able to Reu,qents. cDNAs encoding the human N-formylmethionyl-leucyl- stimulate the AC-II in the presence of activated G,, (Federman et pheiylalanine (fMLP) receptor (in the pCDM8 vecto;) and the-&opidid al., 1992; Tsu et al., 1995a,b; Yung et al., 1995; Chan et al., 1995). recentor (in the DCDMS vector) were urovided bv F. Boulav fLBIO/ Activation of the G,-coupled pathway alone does not stimulate the Lab&atoGe, Greioble, France) and Chris Evans (cniversity oi kalifor- nia, Los Angeles, CA), respectively. The AC-II cDNA was provided by type II activity at all. Recent studies suggest that the G,-proteins Randall Reed (The Johns Hopkins University, Baltimore, MD). The can exert an inhibitory influence on the type II enzyme (Chen and p-adrenergic receptor kinase 495-690 (/3ARK4Y5m(,q0) minigene was a gift Iyengar, 1993). from Robert Lefkowitz (Duke University Medical Center, Chapel Hill, NC). The origin and construction of other cDNAs have been described Received Oct. 12, 1995; revised Nov. 20, 1995; accepted Nov. 28, 1995. previously (Wong et al., 1991, 1992). Pertussis toxin (PTX) was purchased This work was supported by Grants RIG92193SCOS and HKUST 169193 M from from List Biological Laboratories (Campbell, CA). Human embryonic the Universitv and Grants Committee of Hone Kong. We thank Francois Boulav for kidney 293 cells (293 cells hereafter) were obtained from the American ” ” i the human fMLP-receptor cDNA, Chris Evans for the S-opioid receptor, Robert Type Culture Collection (CRL-1573; Rockville, MD). [“H]Adenine was Lefkowitz for the PARK fragment, and Randall Reed for the AC-II. purchased from Amersham (Buckinghamshire, UK). Plasmid purification Correspondence should be addressed to Yung H. Wong at the above address. columns were obtained from Qiagen (Hilden, Germany). Staurosporin Copyright 0 1996 Society for Neuroscience 0270-6474/96/161317-07$05.00/O was purchased from Research Biochemicals (Natick, MA). Cell culture 1318 J. Neurosci., February 15, 1996, 76(4):1317-1323 Tsu and Wong l Stimulation of Type II Adenylyl Cyclase by G, and PMA reagents were obtained from Life Technologies (Gaithersburg, MD), and all other chemicals were purchased from Sigma (St. Louis, MO). Cell culture and transfection. The 293 cells were maintained in Eagle’s minimum essential medium (MEM) supplemented with 10% fetal calf serum (v/v), 50 U/ml penicillin, and 50 pg/ml streptomycin in a humidi- fied atmosphere at 37°C in 5% CO,. COS-7 cells were kept in DMEM containing 10% fetal calf serum. DEAE-dextran-mediated transfection of -zs 5 w 293 and COS-7 cells on 12.well plates has been described previously (Tsu z 0 et al., 1995a). All cDNAs used in the transfection were purified by Qiagen 2 PTX - - + - + - III chromatography. u CAMP accumulation. Twenty-four hours after transfection, 293 cells a,-QL PMA 4-CL-PMA were labeled with [3H]adenine (1 @/ml) in growth medium for 16-20 hr with or without PTX (100 ngiml). The labeled cells were rinsed once with Figure 1. fMLP-induced stimulation of AC-II in the presence of ol,- 2 ml of assay medium (MEM containing 20 mM HEPES, pH 7.4) Q227L or with PMA treatment. The 293 cells were cotransfected with and incubated at 37°C for 30 min with 1 ml of assay medium containing cDNAs encoding the fMLP receptor (0.25 wg/ml) and AC-II (0.25 pg/ml), 1 InM 1-methyl-3-isobutylxanthine and the indicated drugs. Intracellular with or without a,-Q227L (CZ-QL; 0.025 pg/ml). As indicated, transfected [3H]cAMP was isolated by sequential chromatography as described pre- cells were exposed to different conditions before assaying for CAMP viously (Saloman et al., 1974; Wong, 1994). The level of [“HIcAMP was accumulation in response to fMLP (200 nM). Where indicated, PTX (100 estimated by determining the ratios of [3H]cAMP to total [3H]ATP and ngiml) was administered to cells during their labeling with [3H]adenine. [‘H]ADP pools as reported previously (Wong et al., 1991). Absolute Cells were treated with either PMA (100 nM) or I-c-PM (100 nM) for 10 values for CAMP levels varied between experiments, but variability within min before stimulation by fMLP. CAMP accumulation was performed in any single experiment was generally

4 UPMA- + - + - + - + rrx--++- - + + I I

F@rre 3. Requirement of PKC activation in G,-mediated stimulation of AC-II in the absence of activated (Y,.The 293 cells were cotransfected with cDNAs encoding the dopamine D2 receptor (0.25 &ml) and AC-II (0.25 &ml). Where indicated, transfected cells were treated with PTX (100 @ml, 16-20 hr), Sfmrrosporir~ (500 n&t, 30 min), and/or PM4 (100 nM, 10 n Basal q G,,-R agonist min) in sequential order before the addition of dopamine (1 pM, 30 min) q Gi-R agonist &j G$Gq-R agonists for CAMP assays.Data shown are the mean -C SD of triplicate determinations in a single experiment; two independent experiments yielded similar results. Asterisks, Dopamine significantly enhanced the PMA- Figure 4. Synergistic effect of G,,- and G,-coupled receptors on the stimulated AC-II activity; paired t test, p < 0.05. stimulation of AC-II. The 293 cells were cotransfected with cDNAs (0.25 @ml per construct) encoding AC-II, a G,-coupled receptor (ml muscarinic or bombesin), and a G,-coupled receptor (fMLP or dopamine D,) as indicated. Transfected cells were assayed for CAMP accumulation in the presence of one or more agonists to the coexpressed receptors: fMLP (200 a variety of ligands(catecholamines, neuropeptides, or chemotac- nhl), CCh (200 ELM), dopamine (1 PM), and bombesin (100 nbt). Data tic factors) can stimulateAC-II via a G,-linked mechanismthat is shown represent the mean 2 SD of triplicate determinations in one dependenton PKC activation. experiment; two other experiments yielded similar results. Asterisks, Ago- The involvement of PKC wasexamined further with the kinase nists to G,-coupled receptors significantly stimulated the AC-II activity above basal values; paired t test, p < 0.05. Double asterisks, Activation of inhibitor staurosporin. The 293 cells were cotransfected with the G,-coupled receptors significantly potentiated the AC-II responses to cDNAs encodingthe dopamine-D, receptor and AC-II and then agonists acting on the G,-coupled receptors; paired f test, p < 0.05. assayedfor the synergisticeffect of PMA and dopamineon CAMP accumulation.As illustrated in Figure 3, when the transfected cells were pretreated with 500 nM staurosporinfor 30 min before the PMA treatment, the ability of dopamineto stimulate AC-II PMA and G,-coupledreceptors. Other G,-coupled receptorssuch activity was completely abolished.Moreover, the elevated basal as thosefor angiotensinII and cholecystokinin can substitutefor activity that normally accompaniesPMA treatment wasabsent in the ml-muscarinic and bombesin receptors (our unpublished cells pretreated with staurosporin(Fig. 3); staurosporindid not data). affect the basalactivity of AC-II. Collectively, theseresults indi- Stimulation of AC-II by G-protein fly subunits cate that in the absenceof GTP-bound CX$,activation of PKC is required for G,-mediatedstimulation of AC-II. Although not unequivocally proven, it is generally assumedthat the G,-coupled receptors stimulate AC-II via the actions of /3r Synergistic effects of G,- and G,-coupled receptors on subunits(Tang and Gilman, 1991). Stimulation of AC-II by dif- AC-II activity ferent G-protein /3r subunits,in the presenceof activated Gas, If activation of PKC isrequired for the G,-mediatedstimulation of hasbeen well documented(Federman et al., 1992;Taussig et al., AC-II, then agonistsacting on G,-coupled receptorsshould be 1994; Ueda et al., 1994). In all previous reports, GTP-bound (Y, able to mimic the effectsof PMA. We transfected293 cellswith wasabsolutely required for the Pr actionson AC-II. It is therefore cDNAs encoding the AC-II, the ml-muscarinic receptor, and pertinent to examinewhether PMA can replaceactivated CY,and either the fMLP or dopamine-D, receptor. Activation of the permit Pr subunitsto stimulateAC-II. As a control, 293 cellswere ml-muscarinic receptor by 200 ~IVI carbachol (CCh) increased cotransfectedwith cDNAs encodingthe AC-II and cu,-Q227Lin CAMP accumulationby approximately eightfold (Fig. 4). The CCh the absenceor presenceof different combinationsof /3r com- effect has been attributed previously to the phosphorylation of plexes.Coexpression of AC-II and CK,-Q227Lwith either /3irZ or AC-II (Jacobowitz et al., 1993). On the contrary, agonistsacting &y2 in 293cells produced cellsthat exhibit increasedbasal CAMP on the fMLP or dopamine-D, receptorshad little or no effect on accumulation(Fig. 5). In the absenceof a,-Q227L, none of the /3r the activity of AC-II (Fig. 4). In the presenceof 200 PM CCh, both complexesstimulated AC-II (data not shown).However, similar fMLP and dopaminewere able to stimulate AC-II activity, and studiesusing &y2 did not producecells with elevatedCAMP levels the formation of CAMP was elevated by -100% comparedwith (Fig. 5). These resultsare consistentwith previous findingsthat that obtained with CCh alone (Fig. 4). Similar resultswere ob- the former two combinations,but not &y2, were able to yield tained when we replaced the ml-muscarinic receptor with the functional fly complexes(Itiiguez-Lluhi et al., 1992; Pronin and bombesin receptor, another G,-coupled receptor. As shown in Gautam, 1992; Schmidt et al., 1992; Ueda et al., 1994) that can Figure 4, simultaneousactivation of bombesinand dopamine-D, activate AC-II in the presenceof GTP-bound (Y, (Tsu et al., receptorssignificantly increasedCAMP accumulationbeyond that 1995a).To bypassthe requirement of GTP-bound (Y,, 293 cells seenin the presenceof bombesinalone. It is of interest to note coexpressingAC-II and Pr complexeswere incubatedwith 100nbt that the magnitude of synergismbetween G,- and G,-coupled PMA for 10 min before the CAMP assays.Compared with control receptors on the activity of AC-II was similar to that seenwith (cellstransfected with AC-II and vector), the PMA-induced stim- 1320 J. Neurosci., February 15, 1996, 76(4):1317-1323 Tsu and Wong . Stimulation of Type II Adenyiyl Cyclase by G, and PMA

Y PMA 1

Control 01% 02% P3Y2

Figure 5. Coexpression of j+y subunits stimulates AC-II after PMA treatment or in the presence of cr,-Q227L. The 293 cells were cotransfected with the cDNAs encoding AC-II (0.25 &ml) and various combinations of p (0.5 pg/ml) and y (0.25 &ml) subunits, with or without (Y,-Q227L (cu,-QL; 0.01 pg/ml). The cDNAs for the Pr subunits were replaced by equivalent amounts of the vector pcDNA1 in the controls. Basal CAMP accumulation was determined for cells coexpressing (x,- _ -PARK + PARK Q227L. Transfected cells lacking the (Y,-Q227L were treated with PMA n Basal CCh (100 nM for 10 min) before CAMP assays. Results are expressed as a H CCh + dopamine percentage of the CAMP accumulation observed in the Control cells. Data shown represent the mean +- SD of triplicate determinations in a single Figure 7. Blockade of the G,-mediated enhancement of CCh-induced experiment; two other experiments produced similar results. Asterisks, stimulation of AC-II by (Yeand PARK. 293 cells were cotransfected with Significantly different from the corresponding control; paired t test, p < cDNAs (0.25 @ml) encoding AC-II, dopamine-D,, and ml-muscarinic 0.05. receptors with or without (+ or -, respectively) cy,.COS-7 cells were cotransfected as with the 293 cells, except the c~,cDNA was replaced with the @ARK fragment 495-689 (2 CLg/ml). Cells were then labeled with ulation of basal CAMP accumulation was significantly higher in [3H]adenine (1 &i/ml) 1 d before stimulation with CCh (200 p&i) in the cells coexpressing AC-II and either ply2 or p2y2 (Fig. 5). Again, absence or presence of dopamine (1 ELM).Data represent triplicate determinations in a single experiment; hvo independent experiments yielded coexpression of AC-II with pay2 was without effect. Thus, PMA similar results. Asterisks, Significantly higher than the corresponding basal appeared to allow viable /3y subunits to stimulate AC-II, which values; paired t test, p < 0.05. Double asterisks, Dopamine significantly otherwise would require the presence of activated (Y,. enhanced the CCh response; paired t test, p < 0.05. One of the hallmarks of py-mediated signal transduction in mammalian systems is its weaker potency in eliciting the response. Compared with responses mediated by G-protein (Y subunits, a lo-fold higher concentration of j3y subunits might be required value for DPDPE was 6 nhr. This figure closely resembles that for (Lee et al., 1993). For both fMLP and &opioid receptors, the DPDPE-mediated stimulation of phospholipase C (Tsu et al., EC,, values for agonist-dependent, @y-mediated stimulation of 1995b) and is substantially higher than the EC,, of DPDPE- phospholipase C are indeed IO-fold larger than those for induced inhibition of CAMP accumulation. The higher EC,, value cu,-mediated inhibition of adenylyl cyclase (Tsu et al., 1995b,c). supports the notion that the PMA-permissive, Gilinked stimulation of AC-II is mediated via /?r subunits. Using the &opioid receptor as an example, we examined the dose-response relationship between [o-Pen*@-Pen’lenkephalin To ascertain that /3-ysubunits are involved in the G,-mediated (DPDPE) and AC-II stimulation in PMA-treated cells. In 293 stimulation of AC-II after PMA treatment, we exploited the cells coexpressing AC-II and the &opioid receptor, after PMA ability of transducin (a,) and PARK to bind /3r subunits. Both (Y~ and PARK can block py-mediated responses by scavenging free treatment, DPDPE stimulated CAMP accumulation in a dose- /3-ysubunits (Federman et al., 1992; Koch et al., 1994). Cotrans- dependent and saturable manner (Fig. 6). The resultant EC,, fection of 293 cells with AC-II, at, dopamine-D,, and ml- muscarinic receptors produced cells in which dopamine did not potentiate the ability of CCh to stimulate AC-II (Fig. 7). Further- more, coexpression of (Yepartially inhibited the ability of CCh to stimulate CAMP accumulation (Fig. 7). In contrast, dopamine significantly enhanced the CCh response when at was omitted in the transfection (Fig. 7). These results are indicative of the full and partial involvement of /+y subunits in mediating the Gi- and ,.., G,-linked stimulation of AC-II, respectively. SC L. f “.I Using a similar approach, we attempted to demonstrate the -10 -9 -8 -7 -6 ability of PARK,,,-,,, (a truncated form of PARK that has been log IDPDPEI (Ml shown to bind /3r subunits tightly) to prevent the stimulation of AC-II by G,-coupled receptors. In 293 cells, we were unable Figure 6. Agonist dose response for G,-mediated stimulation of AC-II after PMA treatment. The 293 cells were cotransfected with the b-opioid to demonstrate blockade of G,-mediated stimulation of AC-II receptor and AC-II cDNAs as in Figure 2. Transfected cells were treated by P~Km-m, reproducibly. Nevertheless, coexpression of with PMA (100 nM for 10 min) and subsequently assayed for CAMP PARK,,,-,,, with AC-II, the dopamine-D,, and ml-muscarinic accumulation after exposure to varying concentrations of the b-opioid receptors in COS-7 cells caused a loss of dopamine-induced agonist DPDPE (up to 1 FM). Results are expressed as percent maximal stimulation by DPDPE. Data shown represent the mean -C SD of triplicate enhancement of the CCh-stimulated CAMP accumulation (Fig. 7). determinations in a single experiment; hvo additional experiments yielded These results in COS-7 cells are consistent with the idea that by similar results. removing free Py subunits, PARK,,,-,,, effectively abolished the Tsu and Wong l Stimulation of Type II Adenylyl Cyclase by Gi and PMA J. Neurosci., February 15, 1996, 76(4):1317-1323 1321

(A) One Hormone/One Receptor/One G protein (C) One Hormone/Two Receptors/Two G proteins

(B) One Hormone/One Receptor/Two G proteins (D) Two Hormones/Two Receptors/Two G proteins

Figure 8. Mechanistic models in which G,-coupled receptors can activate AC-II in an as-independent manner. Four models (A-D) for the activation of AC-II by G,-released Pr subunits are depicted. H, Hormone; R, receptor; PLCP, phospholipase Cp; PKC, protein kinase C; AC II, type II adenylyl cyclase. ability of G,-coupled receptors to stimulate AC-II. The observed phosphorylation activates the o( subunits of Gi and G,. By the differences between 293 and COS-7 cells are presently undefined. same analogy, loss of function of these phosphorylated (Y subunits might lead to increased CAMP accumulation attributable DISCUSSION to removing the inhibitory control. It has been reported that ai Among the multiple isoforms of adenylyl cyclases cloned to can inhibit AC-II and that this inhibitory effect can be abol- date, the type II and IV isoforms are unique for their positive ished by PKC-mediated phosphotylation (Chen and Iyengar, responsiveness to G-protein P-y subunits. Previous reconstitu- 1993). It is conceivable that an increase in CAMP production by tion (Tang and Gilman, 1991) and transfection (Federman et AC-II could be caused by the removal of inhibitory control by al., 1992) studies have established that the stimulation of AC-II PKC-mediated phosphorylation; but this would only occur if by P-y subunits requires the presence of activated a,. Simulta- there was constitutive suppression of AC-II activity in the neous activation of G, and either Gi or G, would elevate absence of phorbol esters. Moreover, such a mechanism does intracellular CAMP to levels much greater than that seen with not explain the stimulatory effects on AC-II by agonists acting any one of these G-proteins alone. Indeed, activation of Gi- on G,-coupled receptors. The most plausible explanation is that proteins alone does not induce CAMP formation. The AC-II PMA-induced activation of PKC leads to the phosphorylation thus serves as a molecular switch for the integration of coinci- of AC-II, and this modification somehow allows the fly- dent signals (Lust& et al., 1993). The present finding that PMA can alleviate the need for activated (Y, and allow fly subunits to interacting domain of the enzyme to become responsive. Res- stimulate AC-II indicates that the control for AC-II is complex idues 956-982 of AC-II are critical for regulation by By sub- and multifactorial. Although ‘in this scenario stimulation of units (Chen et al., 1995), whereas the putative PKC-responsive AC-II is completely independent of G,, it should be noted that region has been assigned to the C terminus (Levine and Reed, the level of CAMP accumulation observed is only a small 1995). Although the /3y-interacting and the PKC- fraction of that attainable by G, activation (Fig. 1). Thus, even phosphorylation sites are far apart in the primary amino acid though G, is not absolutely essential, it remains one of the most sequence of AC-II, their topology may be significantly closer in potent activators of adenylyl cyclase. the tertiary structure. Only by analyzing the crystal structure of The locus of the PMA action remains to be determined. One AC-II may one be able to resolve this problem. of the defined targets for PKC is AC-II itself. Numerous studies Given that Gi- and G,-regulated pathways can converge on have confirmed that AC-II serves as a PKC substrate and that AC-II, several interesting situations, as depicted in Figure 8, its activity is stimulated after phosphorylation (Jacobwitz et al., may arise. A single receptor that can activate both Gi and G, 1993; Yoshimura and Cooper, 1993; Jacobwitz and Iyengar, should be able to stimulate the activity of AC-II in a partially 1994). Some of the G-protein 01 subunits have also been dem- PTX-sensitive manner. For instance, the thrombin receptor is onstrated to serve as substrates for PKC. Both (Y~(Katada et al., capable of coupling to both Gi- and G,-proteins (Hung et al., 1985) and 01, (Lounsbury et al., 1991) can be phosphorylated by 1992) and receptor activation may lead to the activation of PKC. However, neither o(, nor the By subunits contain poten- PKC, thereby allowing Py subunits released from Gi to stimu- tial phosphotylation sites for PKC. The functional significance late AC-II further (Fig. SB). The a!,-adrenergic receptor is of phosphorylating the o( subunits of Gi and G, remains un- another receptor that is known to possess the ability to interact clear. Because neither (Y~nor (Y, stimulates AC-II (Taussig et with both Gi- and G,-proteins (Conklin et al., 1992). The al., 1994), any gain of function after phosphorylation by PKC is G,-dependent pathway for the activation of PKC can be cir- unlikely to activate the type II isozyme. In fact, one would cumvented by the /3y-sensitive phospholipase C p isoforms expect to see an inhibition of AC-II activity if PKC-mediated (Camps et al., 1992; Smrcka and Sternweis, 1993). Colocaliza- 1322 J. Neurosci., February 15, 1996, 16(4):1317-1323 Tsu and Wong . Stimulation of Type II Adenylyl Cyclase by G, and PMA tion of phospholipase C & or & with AC-II should therefore Preconditioning by PKC-mediated phosphorylation of AC-II allow receptors that are exclusively G,-coupled to stimulate the can dictate whether a signal from a G,-coupled receptor will be formation of CAMP (Fig. SA). However, because the activation transformed into a rise in intracellular CAMP. We predict that of phospholipase C by Pr subunits is much less efficient than many Gi- and G,-coupled receptors can and do produce syn- that by LY~/cY,,, stimulation of AC-II via this mechanism is ergistic effects on AC-II in neuronal tissues. unlikely to produce large increases in intracellular CAMP. 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