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• W2159648646 abstract "The highly conserved topological structure of G protein-activated adenylyl cyclases seems unnecessary because the soluble cytoplasmic domains retain regulatory and catalytic properties. Yet, we previously isolated a constitutively active mutant of theDictyostelium discoideum adenylyl cyclase harboring a single point mutation in the region linking the cytoplasmic and membrane domains (Leu-394). We show here that multiple amino acid substitutions at Leu-394 also display constitutive activity. The constitutive activity of these mutants is not dependent on G proteins or cytosolic regulators, although some of the mutants can be activated to higher levels than wild type. Combining a constitutive mutation such as L394T with K482N, a point mutation that renders the enzyme insensitive to regulators, restores an enzyme with wild type properties of low basal activity and the capacity to be activated by G proteins. Thus regions located outside the cytoplasmic loops of adenylyl cyclases are not only important in the acquisition of an activated conformation, they also have impact on other regions within the catalytic core of the enzyme. The highly conserved topological structure of G protein-activated adenylyl cyclases seems unnecessary because the soluble cytoplasmic domains retain regulatory and catalytic properties. Yet, we previously isolated a constitutively active mutant of theDictyostelium discoideum adenylyl cyclase harboring a single point mutation in the region linking the cytoplasmic and membrane domains (Leu-394). We show here that multiple amino acid substitutions at Leu-394 also display constitutive activity. The constitutive activity of these mutants is not dependent on G proteins or cytosolic regulators, although some of the mutants can be activated to higher levels than wild type. Combining a constitutive mutation such as L394T with K482N, a point mutation that renders the enzyme insensitive to regulators, restores an enzyme with wild type properties of low basal activity and the capacity to be activated by G proteins. Thus regions located outside the cytoplasmic loops of adenylyl cyclases are not only important in the acquisition of an activated conformation, they also have impact on other regions within the catalytic core of the enzyme. adenylyl cyclase expressed during aggregation cytosolic regulator of adenylyl cyclase guanosine 5′-3-O-(thio)triphosphate G protein-coupled adenylyl cyclases are responsible for the synthesis of the ubiquitous second messenger cAMP. In eukaryotic cells, cAMP regulates a multitude of cellular responses, including cell growth and differentiation, metabolism, and synaptic transmission (1Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Google Scholar, 2Simonds W.F. Trends Pharmacol. Sci. 1999; 20: 66-73Google Scholar). Modulation of adenylyl cyclase activity is thus responsible for a wide variety of biological and pathological states. Hormones, neurotransmitters, odorants, and chemokines control this activity by interacting with G protein-coupled receptors. These activated receptors stimulate the exchange of GDP for GTP on the α-subunit of the heterotrimeric G proteins, and the α-subunit dissociates from the βγ complex. Both the activated Gα-subunit and the released Gβγ complex can stimulate or inhibit adenylyl cyclase activity. In mammalian cells, at least nine different forms of this enzyme have been cloned, and each subtype has been shown to possess a specific pattern of regulation and expression. Although these adenylyl cyclases are all activated by Gsα, they show a distinct response to Giα- and Gβγ-subunits, Ca2+, Ca2+/calmodulin, as well as protein kinase A and C. InDrosophila, a large family of these enzymes has also been identified (3Cann M.J. Levin L.R. Adv. Second Messenger Phosphoprotein Res. 1998; 32: 121-135Google Scholar). One enzyme has been shown to be involved in learning and memory (4Levin L.R. Han P.L. Hwang P.M. Feinstein P.G. Davis R.L. Reed R.R. Cell. 1992; 68: 479-489Google Scholar). In the social amoebae Dictyostelium discoideum, the expression of a single G protein-coupled adenylyl cyclase, named ACA,1 is essential for the survival of this lower eukaryote (5Pitt G.S. Milona N. Borleis J.A. Lin K.C. Reed R.R. Devreotes P.N. Cell. 1992; 69: 305-315Google Scholar).Adenylyl cyclases share a common topology predicted to consist of two sets of six transmembrane helices and two large cytoplasmic domains (C1 and C2) located C-terminally to each set of helices (2Simonds W.F. Trends Pharmacol. Sci. 1999; 20: 66-73Google Scholar). Each cytoplasmic domain contains a region of homology (designated C1a and C2a) with the cytoplasmic domains of several adenylyl and guanylyl cyclases. The interaction between the two cytoplasmic domains of adenylyl cyclases is necessary for the activation of the enzyme; the catalytic and G protein-mediated activities are retained by separately purifying and mixing the two cytoplasmic domains (6Tang W.J. Gilman A.G. Science. 1995; 268: 1769-1772Google Scholar). The availability of such a soluble form of adenylyl cyclases has allowed the completion of crystallographic studies (7Zhang G. Liu Y. Ruoha A.E. Hurley J.H. Nature. 1997; 386: 247-253Google Scholar, 8Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Google Scholar). These studies revealed that the C1/C2 catalytic core is a symmetric heterodimer that binds one molecule of Gsα, one molecule of forskolin (a hypotensive drug that stimulates all mammalian forms, except type IX), and one molecule of ATP. The active site of the enzyme is located at the C1/C2 interface. It has been proposed that G proteins regulate the activity of the enzyme by inducing a conformational change that reorients the C1 and C2 domains.In D. discoideum, the proper regulation of the G protein-coupled adenylyl cyclase ACA is essential for the early stages of development during which starvation induces cells to aggregate and differentiate into spores atop a stalk of vacuolated cells (9Parent C.A. Devreotes P.N. Annu. Rev. Biochem. 1996; 65: 411-440Google Scholar). In this system, cAMP acts like a hormone; after its synthesis, it is secreted and it binds to specific G protein-coupled receptors called cARs (cAMP receptors). Receptor occupancy leads to the activation of several effectors including ACA, and the signal is thereby relayed to neighboring cells. Genetic analysis revealed that receptor-mediated activation of ACA requires, in addition to heterotrimeric G proteins, several cytoplasmic regulators. Among them, CRAC (cytosolicregulator of adenylyl cyclase), a novel soluble protein containing a pleckstrin homology domain, is recruited to the plasma membrane in response to receptor activation and is absolutely required for receptor and GTPγS activation of ACA (10Lilly P.J. Devreotes P.N. J. Cell Biol. 1995; 129: 1659-1665Google Scholar). Consequently, crac− cells cannot activate ACA, do not aggregate, and remain as smooth monolayers when starved (11Insall R. Kuspa A. Lilly P.J. Shaulsky G. Levin L.R. Loomis W.F. Devreotes P.N. J. Cell Biol. 1994; 126: 1537-1545Google Scholar). By using random mutagenesis and phenotypic rescue ofcrac− cells, we previously isolated a mutant of ACA displaying constitutive activity that required neither receptor activation nor cytoplasmic regulators (12Parent C.A. Devreotes P.N. J. Biol. Chem. 1996; 271: 18333-18336Google Scholar). This mutant harbored a single point mutation, Leu-394, located N-terminally to the first cytoplasmic domain just after the first hydrophobic cluster. To determine whether this domain is involved in the formation of an activated conformation of ACA, we mutagenized the amino acid sequence in that region and assessed the consequence of these alterations on the developmental and biochemical phenotypes of D. discoideum aca− and crac− cells expressing the mutants.RESULTSBy using site-directed mutagenesis, we replaced Leu-394 with 11 amino acids: 5 non-polar (Ala, Phe, Gly, Ile, and Pro); 3 polar (Asn, Thr, and Tyr); 2 acidic (Asp and Glu); and 1 basic (Arg). These constructs were cloned into an episomal plasmid, downstream of the actin-15 promoter that gives high constitutive levels of expression, and transformed in both aca− andcrac− cells. The developmental phenotype of these cell lines on non-nutrient agar is shown in Fig.1A. All substitutions at Leu-394 gave rise to ACA molecules that could complement theaca− cells, and many of the mutants suppressed the aggregation-deficient phenotype of thecrac− cells. In addition to the original L394S mutation, L394A, L394I, L394G, L394R, and L394T could all aggregate in the absence of the essential cytoplasmic regulator CRAC. One mutant, L394N, differentiated into weak aggregates and was thus scored as a partial suppressor. Western analysis revealed that each mutant expressed similar levels of ACA in both cell lines, although L394P expressed significantly lower levels of protein and was not further analyzed (Fig. 1B). Taken together, these observations suggested that many of the substitutions rendered the enzyme constitutively active.We next evaluated the intrinsic adenylyl cyclase activity of each mutant by measuring the enzyme activity in the presence of Mn2+ (Fig. 2A). Mn2+, a more potent cofactor than Mg2+, will stimulate the enzymatic activity in the absence of G proteins (18Tesmer J.J.G. Sprang S.R. Curr. Opin. Struct. Biol. 1998; 8: 713-719Google Scholar). It is thus used to assess the integrity of the catalytic core. As expected, all mutants expressed in either aca−or crac− cells showed high enzyme activities in the presence of Mn2+ (results are shown for the mutants expressed in crac− cells only). However, their activities, measured in the presence of Mg2+, varied greatly. For a number of mutants, the activity in the absence of Mn2+ was close to their activity in the presence of Mn2+ (Fig. 2B). By comparing the Mg2+/Mn2+ adenylyl cyclase activity ratio of the mutants with their developmental phenotype, we observed that a certain level of adenylyl cyclase activity suppressed the aggregation-deficient phenotype of the crac−cells. A substitution leading to a ratio of 0.5 or greater (corresponding to an activity of ∼50 pmol/min/mg) was sufficient. The glycine mutant defined the boundary between the two classes of mutants. These data show that single point mutations on adenylyl cyclases give rise to enzymes possessing distinct intrinsic activities.Figure 2Adenylyl cyclase activation of Leu-394-substituted ACA mutants expressed incrac− cells.A, basal and MnSO4-stimulated adenylyl cyclase activity. Mutant cell lines were grown, washed, lysed, and assayed for 2 min with and without 5 mm MnSO4 as described under “Experimental Procedures.” The results presented were performed in duplicate and are representative of at least three independent experiments.B, cumulative adenylyl cyclase activation expressed as a ratio of Basal/MnSO4 activity. The values presented are the mean ± S.E. of three to eight independent experiments.L represents the wild type sequence of ACA.View Large Image Figure ViewerDownload (PPT)The original mutant, L394S, rescued the aggregation-deficient phenotype of crac− cells by providing a constant high source of cAMP (12Parent C.A. Devreotes P.N. J. Biol. Chem. 1996; 271: 18333-18336Google Scholar). It displayed no significant activation in response to either receptor or GTPγS stimulation incrac− cells, and the same high basal activity was observed in membranes derived from L394S/gβ− cells, confirming that the mutant was not supersensitive to G proteins or cytosolic regulators. The new mutants showed a similar behavior. As expected, when expressed incrac− cells, none showed a response to GTPγS (data not shown). Next, we transformed three representative mutants ingβ− cells and measured the adenylyl cyclase activity in both lysates and membrane preparations. The three mutants retained high basal activity in the absence of functional G proteins (Fig. 3). Moreover, the high activity was measured in both cell lysates and membrane preparations. Interestingly, for both control and mutant enzymes, the Mg2+/Mn2+ activity ratio was further elevated in membrane preparations compared with cell lysates (Fig. 3). We also observed that many of the constitutively active mutants possessed a residual activation potential because they could be further activated when expressed in aca− cells. In this parental background, where wild type levels of G proteins and CRAC are expressed, the mutants showed a 2–3-fold stimulation in response to GTPγS (Fig. 4). This behavior was also observed with the original L394S mutant. Intriguingly, L394I showed the weakest stimulation in the presence of GTPγS (Fig. 4).Figure 3Adenylyl cyclase activity of Leu-394 substituted ACA mutants expressed ingβ− cells. Basal and MnSO4-stimulated adenylyl cyclase activities were measured in lysates and membrane preparations with and without MnSO4as described under “Experimental Procedures.” The results, performed in duplicates, are expressed as a ratio of Basal/MnSO4 adenylyl cyclase activity and are representative of at least two independent experiments.View Large Image Figure ViewerDownload (PPT)Figure 4GTPγS-stimulated adenylyl cyclase activity in aca− cell lines expressing Leu-394-substituted ACA mutants. Cells were starved for 5 h and assayed for 2 min at room temperature in the presence of 2 mm MgSO4 (basal) or 40 μm GTPγS plus 1 μm cAMP (see “Experimental Procedures”). The results, performed in duplicate, are expressed as a ratio of the adenylyl cyclase activity/MnSO4 activity and are representative of at least two independent experiments.View Large Image Figure ViewerDownload (PPT)We next wanted to more generally disturb the N-terminal domain of the C1 loop of ACA. We generated three mutants that harbored the following alteration in the ACA protein: two mutants were designed to have three alanine insertions either N-terminal (AAAL) or C-terminal (LAAA) from the Leu-394 residue, and one had a 7-amino acid deletion (Del) across the Leu-394 residue, three residues on either side of the leucine along with the Leu-394 residue. Each construct was transformed in bothaca− and crac− cells. Fig. 5A shows that each mutant expressed the mutated ACA protein in both cell lines, although the mutants regularly showed lower expression compared with their wild type counterpart. Phenotypic analysis revealed that none of these substitutions suppressed the aggregation-deficient phenotype of thecrac− cells, suggesting that they did not display constitutive activity (Fig. 5A). When expressed in the aca− background, we observed that the Del mutant acquired a severely impaired conformation. Indeed, although both the AAAL and LAAA mutants could complement theaca− cells, the Del/aca− cell lines remained aggregation-deficient when plated on non-nutrient agar (Fig.5A). These results suggest that this mutation has a deleterious effect on G protein-mediated activation of ACA. Indeed, as shown in Fig. 5B, the Del/aca−showed only a weak response to GTPγS. It thus appears that the N-terminal domain of the C1 loop of adenylyl cyclases is critical for the acquisition of an activated conformation.Figure 5Adenylyl cyclase activity of wild type, alanine insertional, and deletion ACA mutants.A, ACA protein expression and developmental phenotypes of the mutants when expressed in aca− andcrac− cells. B, adenylyl cyclase activity was performed as described in legend of Fig. 4. The results, performed in duplicates, are expressed as a ratio of the adenylyl cyclase activity/MnSO4 activity and are representative of at least five independent experiments. L represents the wild type sequence of ACA. LAAA and AAAL designate 3′ and 5′ of Leu-394 alanine insertions. Del indicates a mutant of ACA carrying a 7-amino acid deletion spanning the Leu-394 residue.View Large Image Figure ViewerDownload (PPT)To address this point further, we designed an enzyme that contained, in addition to a constitutive mutation, a substitution that rendered the enzyme insensitive to G protein activation. In a previous screen designed to isolate loss-of-function mutants of ACA, we identified several mutants that remained aggregation-deficient when expressed inaca− cells. These mutants displayed normal Mg2+/Mn2+ adenylyl cyclase activity ratios but showed no response to GTPγS stimulation and were unable to enter development when plated on non-nutrient agar (16Parent C.A. Deveotes P.N. J. Biol. Chem. 1995; 270: 22693-22696Google Scholar). One of these uncoupled mutants harbored a single point mutation (K482N) in the C1 loop of ACA. We introduced the L394T substitution, the strongest constitutive mutant, in the K482N background to construct the double mutant. The double mutant was electroporated inaca− cells, and the developmental phenotype of the resulting transformants was assessed. Western analysis revealed that the L394T,K482N/aca−, and the Leu-394/aca− cells expressed similar levels of ACA (data not shown). Under normal plating conditions, the mutant cells did not rescue the aggregation-deficient phenotype of theaca− cells. However, when plated at higher density, the L394T,K482N/aca− cells did enter development to form small mounds and fruiting bodies, a result never observed with aca− or K482N/aca− cells (Fig.6A). The presence of K482N altered the basal activity of the L394T mutation. Under our standard measurement conditions, L394T,K482N displayed a Mg2+/Mn2+ adenylyl cyclase activity ratio of 0.3 compared with > 0.7 for the L394T mutant. On the other hand, L394T,K482N exhibited a significant activation in the presence of GTPγS (Fig. 6B). This activation, which was never observed in K482N/aca− cells, was also detected when the adenylyl cyclase activity was measured in vivo after receptor stimulation (Fig. 6C).Figure 6Developmental and biochemical phenotype of L394T,K482N/aca− cells.A, aca− and L394T,K482N/aca− cells were grown in liquid culture and washed, and 1 × 108 cells were plated on non-nutrient agar on 35-mm plates at 22 °C. Photographs were taken 24 h after the cells were plated. B, basal, MnSO4, and GTPγS-stimulated adenylyl cyclase activity in L/aca−, K482N/aca−, and L394T,K482N/aca− cells. The assays were performed as described under “Experimental Procedures.” The results were performed in duplicate and are representative of at least three independent experiments. C, receptor-mediated activation of adenylyl cyclase in L/aca−, K482N/aca−, and L394T,K482N/aca− cells. Cells were stimulated with 10 μm cAMP, rapidly lysed at specific time points, and assayed as described under “Experimental Procedures.” The results were performed in duplicate and are representative of at least two independent experiments.View Large Image Figure ViewerDownload (PPT)DISCUSSIONThroughout evolution, the topology of G protein-coupled adenylyl cyclases has been remarkably conserved. D. discoideum,Drosophila, and mammals all express enzymes that are predicted to display two sets of 6 transmembrane domains followed by a large cytoplasmic loop (Fig. 7). Although it has been proposed that some adenylyl cyclases are responsive to transmembrane potential, the exact function of the transmembrane spans has remained largely unknown (19Reddy R. Smith D. Wayman G. Wu Z. Villacres E.C. Storm D.R. J. Biol. Chem. 1995; 270: 14340-14346Google Scholar). Our data show that regions linking the transmembrane domains to the cytoplasmic loops are important for proper regulation of the enzyme. The crystal structure of the soluble adenylyl cyclase with Gsα revealed that the α-subunit of G proteins binds to a crevice on the outside of the C2 loop on residues mainly located on the α2 helix, as well as to residues on the N-terminal portion of the C1 loop (8Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Google Scholar). The Leu-394 residue of ACA that is mutated in this study is located ∼35 amino acids upstream of this N-terminal portion of the C1 loop (Fig. 7). In fact, important biochemical differences between the soluble and native forms of adenylyl cyclases have been observed. First, the affinity of the soluble adenylyl cyclase for Gsα is significantly reduced, ∼50-fold, compared with the native enzyme (20Tang W.J. Krupinski J. Gilman A.G. J. Biol. Chem. 1991; 266: 8595-8603Google Scholar, 21Sunahara R.K. Dessauer C.W. Whisnant R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: 22265-22271Google Scholar). Second, the effects of Gβγ-subunits on the chimeric soluble preparations do not recapitulate what has been observed with the purified full-length enzymes (22Taussig R. Quarmby L.M. Gilman A.G. J. Biol. Chem. 1993; 268: 9-12Google Scholar, 23Dessauer C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Google Scholar). Finally, the maximal stimulated activity is ∼10-fold higher for the soluble enzyme (21Sunahara R.K. Dessauer C.W. Whisnant R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: 22265-22271Google Scholar, 22Taussig R. Quarmby L.M. Gilman A.G. J. Biol. Chem. 1993; 268: 9-12Google Scholar). Moreover, the F400Y mutation in the type V molecule, which showed increased basal activity and sensitivity to Gsα and forskolin, did not alter the enzymatic activity of the soluble construct bearing the same substitution (24Zimmermann G. Zhou D. Taussig R. Mol. Pharmacol. 1999; 56: 895-901Google Scholar, 25Hatley M.E. Benton B.K. Xu J. Manfredi J.P. Gilman A.G. Sunahara R.K. J. Biol. Chem. 2000; 275: 38626-38632Google Scholar). It has been shown that the C1b domain of adenylyl cyclases (which is absent in these constructs) possesses regulatory properties (26Dessauer C.W. Tesmer J.J.G. Sprang S.R. Gilman A.G. J. Biol. Chem. 1998; 273: 25831-25839Google Scholar, 27Scholich K. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2915-2920Google Scholar, 28Scholich K. Wittpoth C. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9602-9607Google Scholar, 29Tan C.M. Kelvin D.J. Litchfield D.W. Ferguson S.S.G. Feldman R.D. Biochemistry. 2001; 40: 1702-1709Google Scholar, 30Wittpoth C. Scholich K. Yigzaw Y. Stringfield T.M. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9551-9556Google Scholar, 31Yan S. Beeler J. Chen Y. Shelton R. Tang W. J. Biol. Chem. 2001; 276: 8500-8506Google Scholar). Particularly, the type VI enzyme has a cAMP-dependent protein kinase phosphorylation site within its C1b region that dampens Gsα-mediated activation (32Chen Y. Harry A. Li J. Smit M.J. Bai X. Magnusson R. Pieroni J.P. Weng G. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14100-14104Google Scholar). In addition, glycosylation of the extracellular domains of the type VI enzyme has recently been shown to be important for catalytic activity (33Wu G.C. Lai H.L. Lin Y.W. Chu Y.T. Chern Y. J. Biol. Chem. 2001; 276: 35450-35457Google Scholar). It is thus possible that some of the differences observed between the native and soluble forms of adenylyl cyclases could be explained by the lack of these domains in the soluble constructs. Our data support and extend these observations by defining yet another region of the native structure that is critical for activation.Figure 7Sequence analysis of the location of the ACA mutants. The C1 and C2 domains of D. discoideumACA and the mammalian type II adenylyl cyclases were aligned using ClustalW analysis. The amino acid position of the sequences is shown at the end of each line. The secondary structure of the C1 loop (8Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Google Scholar) is drawn below the consensus sequence inblue. The Leu-394 and Lys-482 residues are shown inred and blue, respectively. The L405S and F421S mutations are shown in green. The putative native structure of adenylyl cyclases is illustrated in the bottom left corner. The boxed region represents the sequence contained within the soluble construct.View Large Image Figure ViewerDownload (PPT)Hatley et al. (25Hatley M.E. Benton B.K. Xu J. Manfredi J.P. Gilman A.G. Sunahara R.K. J. Biol. Chem. 2000; 275: 38626-38632Google Scholar) isolated mutants of the type II enzyme that rescued the cyclase-null Saccharomyces cerevisiaestrain. All mutations mapped to the cytoplasmic loops of the enzyme. To investigate the biochemical defect of the mutants, the substitutions were engineered in the type V C1/type II C2 soluble construct. Of 13 mutants analyzed in this context, only a few displayed substantial changes in adenylyl cyclase activity when compared with the wild type control. Because the activity of the mutants was not measured in the context of the native type II enzyme, the lack of change in basal activity in most of the mutants analyzed may be due to the absence of the transmembrane domains or, alternatively, to the supersensitivity of the yeast screen.We have shown that the activity of native adenylyl cyclases can be dramatically modulated by substituting Leu-394 of ACA. This residue is conserved in eight of nine of the mammalian adenylyl cyclases (Fig. 7) (12Parent C.A. Devreotes P.N. J. Biol. Chem. 1996; 271: 18333-18336Google Scholar). Substituting Leu-394 of ACA with a variety of other residues gives rise to mutants possessing high basal activity. Intriguingly, the substitutions lead to graded ranges of high basal activity suggesting that each mutant acquires a conformation that progressively reproduces the active state of the enzyme and that the Leu-394 position is critical in the formation of an activated conformation of adenylyl cyclases. This type of graded effect has also been observed when the Ala-293 position of the α1B-adrenergic receptor is mutated to other amino acids (34Kjelsberg M.A. Cotecchia S. Ostrowski J. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1430-1433Google Scholar). A closer analysis of the nature of the amino acids leading to the broad spectrum of intrinsic adenylyl cyclase activation reveals no particular allegiance regarding charge, size, or hydrophobicity. Surprisingly, the subtlest mutation (L394I) provides significant constitutive activity. It thus seems that changes in the local conformation is responsible for the acquired high activity. Consequently, we do not expect that substitutions of this particular residue in other adenylyl cyclases will lead to constitutively active enzymes. Indeed, the mammalian type V enzyme harbors a serine at the corresponding Leu-394 position and does not display unusually high basal activity. We propose that changes in the N terminus region of the C1 loop will lead to enzymes that display high basal activity. A screen similar to the one we developed for ACA will be required to identify such mutations in mammalian enzymes (12Parent C.A. Devreotes P.N. J. Biol. Chem. 1996; 271: 18333-18336Google Scholar).Mutations localized to the N terminus of the C1 loop of ACA also give rise to enzymes that are non-responsive to G proteins. In a previous study, we isolated a mutant that harbored two point mutations (L405S and F421S) adjacent to the Leu-394 position (Fig. 7). This mutant showed normal enzymatic activity in the presence of MnSO4. However, it was devoid of any G protein-mediated activation (16Parent C.A. Deveotes P.N. J. Biol. Chem. 1995; 270: 22693-22696Google Scholar). This behavior is identical to the one observed with the Del mutant engineered in the present study. Interestingly, the AAAL mutant had the opposite phenotype displaying increased G protein-mediated activation compared with the wild type enzyme. Taken together, these mutants, which all harbor mutations in a discrete region outside the soluble domains, underscore the importance of this region on the activation potential of adenylyl cyclases.Structural information shows that the C1/C2 heterodimer structure is based on highly organized interactions between the two domains. It has been suggested that extensive hydrogen bonding between the β4-β5 loops of C2 and the β2-β3 loops of C1 exist and that alterations in these interactions have deleterious effects on enzymatic activity (8Tesmer J.J.G. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Google Scholar). The ACA K482N mutant, which is specifically devoid of G protein-stimulated activity, harbors a point mutation in the highly conserved β2 loop of C1 (Fig. 7). Mutations of this exact residue in mammalian adenylyl cyclases also give rise to defective enzymes (35Tang W.J. Stanzel M. Gilman A.G. Biochemistry. 1995; 34: 14563-14572Google Scholar,36Zimmermann G. Zhou D. Taussig R. J. Biol. Chem. 1998; 273: 19650-19655Google Scholar). The addition of the constitutive mutation L394T to the K482N substitution biochemically and phenotypically suppresses the defects of the K482N mutant. The L394T,K482N/aca− cells regain the capacity to aggregate, respond to GTPγS, and show a significant response to receptor stimulation. As with the original K482N/aca− mutant, these results again show that the Lys-482 residue is not essential for the catalytic activation of adenylyl cyclases. The fact that the constitutive mutation can suppress the loss-of-function defect shows that the two sites, although distant, influence each other. Intriguingly, whereas the double mutant regains the capacity to be activated by G proteins, it does not exhibit the constitutive activity of the L394T mutant. It thus appears that the hig" @default.
• W2159648646 created "2016-06-24" @default.
• W2159648646 creator A5010410867 @default.
• W2159648646 creator A5066573853 @default.
• W2159648646 creator A5091366547 @default.
• W2159648646 date "2002-01-01" @default.
• W2159648646 modified "2023-09-30" @default.
• W2159648646 title "Regulation of Adenylyl Cyclases by a Region Outside the Minimally Functional Cytoplasmic Domains" @default.
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• W2159648646 doi "https://doi.org/10.1074/jbc.m106430200" @default.
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