Cell, Vol. 70, 669-672,
September
16, 1992, Copyright
0 1992 by Cell Press
Adenylyl Cyclases
Minireview
WeiJen Tang and Alfred G. Gilman Department of Pharmacology University of Texas Southwestern Medical Center Dallas, Texas 75235
The activities of many adenylyl cyclases, the enzymes that convert ATP to cyclic AMP, are controlled dynamically by a variety of hormones, neurotransmitters, and other regulatory molecules. As a result, this reaction is dominant among those used to modulate intracellular concentrations of cyclic AMP. The cyclic nucleotide, in turn, activates several target molecules-primarily cyclic AMP-dependent protein kinases- to control such diverse phenomena as metabolism, gene transcription, and memory. Although adenylyl cyclases have been studied for more than three decades, most of the detailed molecular information about the enzymes of higher eukaryotes has been acquired only recently, following the cloning of a cDNA encoding one of these proteins (Krupinski et al., 1989). Divergent and Unexpected Structures Cyclic AMP is used universally as a signaling molecule, and adenylyl cyclases are incorporated into a variety of protein structures. Most adenylyl cyclases are associated with the plasma membrane, although certain bacterial enzymes (and perhaps one form in mammalian sperm) are cytosolic. There are at least three general classes of mem-
Adenylyl Type
Ml
Cyclases:
brane-bound adenylyl cyclases (Figure 1). Peripheral membrane proteins are found in Saccharomyces cerevisiae and Escherichia coli, while one form of the enzyme in Dictyostelium appears to have a single transmembrane span. The most common motif in higher eukaryotes includes a short amino-terminal region (N) and two -40 kd cytoplasmic domains (C, and C,), punctuated by two intensely hydrophobic stretches (MI and M2); each of the latter is hypothesized to contain six transmembrane helices. It is this complex and widely distributed group of adenylyl cyclases that is responsive to stimulatory and inhibitory regulation by hormones and neurotransmitters, acting via the intermediacy of both receptors (generally with seven transmembrane spans) and heterotrimeric guanine nucleotide-binding regulatory proteins or G proteins (Gilman, 1987). cDNAs encoding eight adenylyl cyclases of this general type have now been cloned: six of these (arbitrarily designated types I-VI) are from mammalian sources, one is the product of the rutabaga gene from Drosophila, and one is from Dictyostelium. Among these eight adenylyl cyclases, portions of the C, and C, domains (designated C,. and CL) are well conserved (50%-92% sequence identity among the mammalian enzymes). Of interest, C,, and Cna are also very similar to each other and to the catalytic domains of a series of membrane-bound guanylyl cyclases (Chinkers and Garbers, 1991). The catalytic activity of these adenylyl cyclases
M2
Figure 1. Structures of Eukaryotic Cyclases and Guanylyl Cyclases
See text for description. Bold lines indicate regions of similarity; the hatched region in the adenylyl cyclase from S. cerevisiae has low but detectable homology with the domains shown as bold lines. A, ATP-binding regulatory domain in the membrane-bound guanylyl cyclases.
I-VI
Drosophila Rutabaga Dictyostelium ACA
n
Dictyostelium ACG Adenylyl Cyclase
Adenylyl
n
S. cerevisiae Adenylyl
Cyclase
Membrane Bound Guanylyl Cyclases
Soluble Guanylyl Cyclases
Cell 870
requires both C,, and CPa and may be regulated by their interaction. Thus, several point mutations in either C,, or C& compromise catalytic activity severely (Tang et al., 1992). Further, the amino-terminal and carboxyl-terminal halves of type I adenylyl cyclase have no catalytic activity when expressed alone, but regulated enzymatic activity is restored when they are expressed concurrently (Tang et al., 1991). Analogously, soluble forms of guanylyl cyclase are heterodimers, and each subunit contains a region that is homologous to C,, and CZa; both subunits are required for catalysis (Nakane et al., 1990). Membrane-bound guanylyl cyclases may also function as dimers or higher oligomers. The locations of the nucleotide-binding site(s) within C,, and COa are not yet known, and there are no obvious sequence homologies with other nucleotide-binding proteins. However, adenylyl cyclases are inhibited (noncompetitively with respect to Mg2+/ATP) by adenosine, 3’-AMP, and related analogs (so-called P-site inhibitors). The most obvious suggestion is that C,, and Cna represent, individually, domains for interaction with substrate and P-site inhibitors. However, the studies mentioned above suggest that both domains might contribute to the binding of both types of molecule. The apparent molecular weight of brain adenylyl cyclase (a mixture of forms) has been estimated to be 200-250 kd, based on hydrodynamic properties (Neer et al., 1980). By contrast, the actual molecular weights of the various species of adenylyl cyclase now known to be expressed in brain approximate 120 kd. Perhaps adenylyl cyclase is a dimer (actually a tetramer of Cl,- and &,-like domains). If so, catalytically inactive point mutations in C,, would be expected to complement those in C&, but they do not. It is perhaps more likely that the large apparent molecular weight of the unpurified enzyme reflects contributions from associated proteins (particularly G proteins and perhaps receptors or unidentified players). Amino acid sequence similarities among the various adenylyl cyclases are limited beyond the C,, and CZ, domains. However, as mentioned, the overall structure is well conserved from Dictyostelium to mammals-raising the question of the function of the two sets of six transmembrane spans (other than to organize or orient the two cytoplasmic structures). The extracellular domains might serve a receptor-like role, but there is little evidence for such and the amount of extracellular structure is probably small. The hydrophobic domains are presumed to be the site for activation by forskolin, a lipid-soluble diterpene. Perhaps there are endogenous counterparts in mammalian cells. It has been hypothesized that adenylyl cyclase might have a dual life, serving as a channel or transporter as well (Krupinski et al., 1989). This notion was based almost entirely on the unique topographical homology between adenylyl cyclase and various ion channels and transporters (particularly the P glycoprotein and the cystic fibrosis transmembrane conductance regulator). However, there are no sequence homologies between the adenylyl cyclases and any of the channels or transporters. Many cells secrete cyclic AMP, and Dictyostelium utilizes the cyclic
nucleotide as a “hormone” to control Cellular aggregation and differentiation. It might be reasonable to couple synthesis of cyclic AMP with its secretion by incorporating both functions into one molecule. Dictyostelium synthesizes two adenylyl cyclases-one that resembles the mammalian enzymes (AC-A) and one with a single transmembrane span (AC-G). Cyclic AMP secretion is normal when only AC-G is expressed, effectively ruling out this hypothesis (Pitt et al., 1992). Of interest, an adenylyl cyclase from Paramecium has associated pore-forming activity with selectivity for cations (observed after reconstitution into lipid bilayers) (Schultzet al., 1992). Unfortunately, the structure of this enzyme is not known. Channel activity has not been detected when mammalian adenylyl cyclases have been expressed in insect (Sf9) cells. The diversity of amino acid sequences among the hydrophobic domains of the various adenylyl cyclases would also seem to argue against their role as channels or transporters. Diverse Regulatory Properties The diverse structures of the adenylyl cyclases mirror the wide range of regulatory influences that impinge on them. At one extreme, the relatively simple soluble enzyme from Streptococcus salivarius is dependent on a-keto acids for activity. The mammalian enzymes represent the opposite pole, with type-specific stimulatory and inhibitory regulation by G protein a and by subunits, Ca*+-calmodulin, P-site inhibitors, and forskolin. One aspect of the evolution of these regulatory properties is particularly striking. The adenylyl cyclase from Saccharomyces cerevisiae is stimulated by a GTP-binding protein, as are the mammalian enzymes. Thus, this important regulatory motif is established in a primitive organism. However, the molecular players bear little relationship to one another. The stimulatory GTP-binding protein in yeast is ras, despite the fact that yeast have heterotrimeric G proteins, and the cyclase is a 230 kd peripheral membrane protein with only the slightest resemblance to the mammalian adenylyl cyclases (Kataoka et al., 1985). The properties of the eukaryotic adenylyl cyclases that have been cloned to date are presented in Table 1. All the mammalian enzymes are activated by the a subunit of G,, the G protein first identified because of this action. Some (particularly type I) are also activated by Ca2+-calmodulin. Certain enzymes are rather broadly expressed, while others are very restricted in their distribution (particularly type Ill) (Sakalyar and Reed, 1990). One cloned adenylyl cyclase from Drosophila (the product of the rutabaga gene) is similar biochemically to the mammalian type I enzyme. However, the molecule is remarkable in that it has a very long (110 kd) carboxyl-terminal extension of unknown significance. A missense mutation in the rutabaga adenylyl cyclase destroys catalytic activity and is associated with a learning deficiency in the fly (Levin et al., 1992). The adenylyl cyclases (from murine S49 cells and platelets) that were originally characterized with regard to the effects of G proteins are activated by G,, and are largely insensitive to the G protein by subunit complex. Similar results with other G protein-regulated effecters (particularly retinal cyclic GMP phosphodiesterase) led to the concept that G protein a subunits regulate effecters, while pr
Minireview 071
Table
1. Properties
of Eukaryotic
Adenylyl
Cyclases Effect
Type” I II III IV V VI rutabaga AC-A AC-G CYRl
Amino
Acid
Residues
1134 1090 1144 1964 1184 1165 2249 1407 658
of G Proteins
Expression
Pr
Brain Brain, lung Olfactory Brain, others Heart, brain, others Heart, brain, others Mushroom body During aggregation Fruiting body Constitutive
+ 0 + 0 0 0 ? ?d 0
Ca2+-Calmodulin
Forskolin
+ 0 + 0 0 0 + ? ?d 0
+ + + t t + + 0 0 0
B Adenylyl cyclase types I-VI are mammalian; rutabaga is from Drosophila; AC-A and AC-G are from Dictyostelium; CYRI is from Saccharomyces cerevisiae. References: I (Krupinski et al., 1989) II (Feinstein et al., 1991) Ill (Sakalyar and Reed, 1990) IV (Gao and Gilman, 1991) V (Ishikawa et al., 1992) VI (Premont et al., 1992; Katsushika et al., 1992) rutabaga (Levin et al., 1992) AC-A and AC-G (Pitt et al., 1992) and CYRl (Kataoka et al., 1985). b +, stimulates; -, inhibits; 0, no effect. c AC-A is activated by GTPTS; however, a homolog of G,a has not been detected in Dictyostelium. d Unlikely to have an effect; GTPyS does not activate. n Activated bv ras.
was assigned less interesting roles. Therefore, when the novel adenylyl cyclase clones were expressed and examined individually, it was surprising to detect prominent, type-specific regulation of enzymatic activity by by. Whereas 9~ inhibits type I adenylyl cyclase when stimulated by either G., or Ca2+-calmodulin, fir greatly potentiates the stimulatory effect of G,, on either type II or type IV adenylyl cyclase (Tang and Gilman, 1991). The other adenylyl cyclases cloned to date are relatively unaffected by Pr. The concentration of by required to modulate adenylyl cyclase activity is significantly greater than that of G,a, and thus it appears that the G. oligomer would not be the source of 9~ for such regulation. (Besides, it is unclear what the point would be of simultaneously stimulating and
inhibiting type I adenylyl cyclase by dissociation of the activated subunits of G,). Rather, it is assumed that the necessary concentrations of S+r are contributed by G proteins such as Gi and G,, which are far more abundant than G, (at least in brain). This provides a clear mechanism for cross-talk and interaction between signaling pathways. Adenylyl cyclase is activated by G,-linked pathways but can be inhibited or stimulated further by 9~ when other G protein-mediated pathways are activated simultaneously (Figure 2). Such conditional, synergistic activation of type II adenylyl cyclase by G,- and Gi-mediated pathways has been demonstrated in intact cells by transient expression of cDNAsencoding the requisite proteins(Federman et al., 1992). This mechanism may well underlie the synergistic effects of neurotransmitters on cyclic AMP accumulation
Kinase (?) Figure
2. Regulation
of Mammalian
Adenylyl
Cyclases
See text for description. R. G protein-coupled receptors; G protein a subunit; CaM, calmodulin; 3’-AMP, a P-site
G, and G,, G proteins inhibitor.
that stimulate
and inhibit adenylyl
cyclase,
respectively;
Ga, any
Cdl 872
in brain slices, which were documented 20 years ago (Sattin et al., 1975). Regulation of effecters by G protein 6~ subumts may well be a general phenomenon. Although the effects of 67 on K+ channels remain controversial, 67 has recently been shown to activate one form of phospholipase C from HL60 cells (Camps et al., 1992). The question of specificity then arises, since four isoforms of 9 and an increasing number of distinctrsubunits have been identified. Defined 6~ complexes can be assembled by expression in Sf9 cells and purified facilely by subunit affinity chromatography. There is some specificity in their interactions with adenylyl cyclase, and this interaction is dependent on prenylation of the G protein y subunit (IAiguez-Lluhi et al., 1992). Receptors that interact with members of the Gi family of G proteins (Gil, G,2, and Gis) mediate inhibition of adenylyl cyclase. Although the G, proteins are demonstrably important (revealed by reconstitution of membranes with Gi oligomers), mechanisms are uncertain and may vary with the type of cyclase in question. Transfection of cells with constitutively active mutant forms of G,a results in inhibition of cyclic AMP accumulation, implicating a subunits in the pathway (Wong et al., 1992). However, activated Gia subunits are very weak inhibitors (at best) of adenylyl cyclase when tested in vitro-in membranes or with purified cyclases. Perhaps Gia proteins inhibit adenyfyl cyclase indirectly by a pathway that remains to be reconstituted in vitro. However, when G protein 6~ subunits are tested in vitro. inhibitory effects are observed readily. This effect is y on type I adenylyl cyclase. The effect is her cyclases and is apparently due to deactiry f3r (Gilman, 1964). Although the effort that understanding mechanisms of inhibition of adenylyl cyclase by the G, proteins may exceed the physiological significance of the phenomenon, the solution of this problem will almost certainly yield valuable insights into the scope and mechanism of action of this important group of G proteins. References Bakalyar, H. A. and Reed, R. R. (1990). Science 250, 1403-1406 Camps, M., Hou, C. F., Sidiropoulos, D., Stock, J. B., Jakobs, K. H., and Gierschik, P. (1992). Eur. J. Biochem. 206, 821-831. Chinkers. M. and Garbers, D. L. (1991). Annu. Rev. Biochem. 60,553575. Federman, A. D., Conklin, B. R., Schrader, Bourne, H. R. (1992). Nature 356, 159-161.
K. A., Reed,
Ft. R., and
Feinstein, P. G., Schrader, K. A., Bakalyar. H. A., Tang, W.-J., Krupinski, J., Gilman, A. G., and Reed, R. R. (1991). Proc. Natl. Acad. Sci. USA 88, 10173-10177. Gao, B., and Gilman, 10178-10162.
A. G. (1991).
Proc.
Gilman,
A. G. (1964).
Cell 36, 577-579.
Gilman,
A. G. (1987).
Annu.
Rev. Biochem.
Natl. Acad.
Sci. USA 88,
56, 615-649
Iriiguez-Lluhi, J. A., Simon, M. I., Robishaw, J. D., and Gilman, A. G. (1992). J. Biol. Chem., in press. Ishikawa, Y.. Katsushika, S., Chen, L., Halnon, N. J., Kawabe, J.-i., and Homey, C. J. (1992). J. Biol. Chem. 267, 13553-13557. Kataoka,
T., Broek,
D., and Wigler,
M. (1985).
Cell 43, 493-505.
Katsushika. S., Chen, L., Kawabe, J.-i., Nilakantan, R., Halnon, N. J., Homey, C. J.. and Ishikawa, Y. (1992). Proc. Natl. Acad. Sci. USA, in press.
Krupinski, J., Coussen, F., Bakalyar, H. A., Tang, W.-J., Feinstein, P. G., Orth, K., Slaughter, C., Reed, R. R., and Gilman. A. G. (1989). Science 244, 1558-1564. Levin, L. R., Han, P.-L., Hwang, P. M., Feinstein, and Reed, R. R. (1992). Cell 68, 479-489.
P. G., Davis,
Nakane, M., Arai, K., Saheki, S., Kuno. T., Buechler, F. (1990). J. Biol. Chem. 265, 16841-16845. Neer, E. J., Echeverria, 9782-9789.
D., and Knox,
S. (1980).
Sattin, A., Rail, T. W., and Zanella, 192, 22-32. Schultz, Schmid, Tang,
J. (1975).
W., and Murad,
J. Biol. Chem.
Pitt, G. S., Milona, N., Borleis, J., Lin, K. C., Reed, reotes, P. N. (1992). Cell 69, 305-315. Premont, R. T., Chen, J., Ma, H.-W., Ponnapalli, (1992). Proc. Natl. Acad. Sci. USA, in press.
W.-J. and Gilman,
A. G. (1991).
M., and lyengar,
J. Pharmacol.
Tang, W.-J., Iriiguez-Lluhi, (1992). Cold Spring Harbor Wang, Y. H.. Conklin, 339-341.
Science
J., and Gifman, J. A., Mumby, Symp. &ant.
B. R., and Bourne,
255,
R. R., and Dev-
J. E., Klumpp, S., Benz, R., Schurhoff-Goeters. A. (1992). Science 255, 600-603.
Tang, W.-J., Krupinski, 266, 8595-8603.
R. L.,
R.
Exp. Ther. W. J. C., and
254, 1500-1503.
A. G. (1991).
J. Biol. Chem.
S. M., and Gilman, Biol. 57, in press. H. R. (1992).
Science
A. G. 255,
September
16, 1992, Copyright
0 1992 by Cell Press
Adenylyl Cyclases
Minireview
WeiJen Tang and Alfred G. Gilman Department of Pharmacology University of Texas Southwestern Medical Center Dallas, Texas 75235
The activities of many adenylyl cyclases, the enzymes that convert ATP to cyclic AMP, are controlled dynamically by a variety of hormones, neurotransmitters, and other regulatory molecules. As a result, this reaction is dominant among those used to modulate intracellular concentrations of cyclic AMP. The cyclic nucleotide, in turn, activates several target molecules-primarily cyclic AMP-dependent protein kinases- to control such diverse phenomena as metabolism, gene transcription, and memory. Although adenylyl cyclases have been studied for more than three decades, most of the detailed molecular information about the enzymes of higher eukaryotes has been acquired only recently, following the cloning of a cDNA encoding one of these proteins (Krupinski et al., 1989). Divergent and Unexpected Structures Cyclic AMP is used universally as a signaling molecule, and adenylyl cyclases are incorporated into a variety of protein structures. Most adenylyl cyclases are associated with the plasma membrane, although certain bacterial enzymes (and perhaps one form in mammalian sperm) are cytosolic. There are at least three general classes of mem-
Adenylyl Type
Ml
Cyclases:
brane-bound adenylyl cyclases (Figure 1). Peripheral membrane proteins are found in Saccharomyces cerevisiae and Escherichia coli, while one form of the enzyme in Dictyostelium appears to have a single transmembrane span. The most common motif in higher eukaryotes includes a short amino-terminal region (N) and two -40 kd cytoplasmic domains (C, and C,), punctuated by two intensely hydrophobic stretches (MI and M2); each of the latter is hypothesized to contain six transmembrane helices. It is this complex and widely distributed group of adenylyl cyclases that is responsive to stimulatory and inhibitory regulation by hormones and neurotransmitters, acting via the intermediacy of both receptors (generally with seven transmembrane spans) and heterotrimeric guanine nucleotide-binding regulatory proteins or G proteins (Gilman, 1987). cDNAs encoding eight adenylyl cyclases of this general type have now been cloned: six of these (arbitrarily designated types I-VI) are from mammalian sources, one is the product of the rutabaga gene from Drosophila, and one is from Dictyostelium. Among these eight adenylyl cyclases, portions of the C, and C, domains (designated C,. and CL) are well conserved (50%-92% sequence identity among the mammalian enzymes). Of interest, C,, and Cna are also very similar to each other and to the catalytic domains of a series of membrane-bound guanylyl cyclases (Chinkers and Garbers, 1991). The catalytic activity of these adenylyl cyclases
M2
Figure 1. Structures of Eukaryotic Cyclases and Guanylyl Cyclases
See text for description. Bold lines indicate regions of similarity; the hatched region in the adenylyl cyclase from S. cerevisiae has low but detectable homology with the domains shown as bold lines. A, ATP-binding regulatory domain in the membrane-bound guanylyl cyclases.
I-VI
Drosophila Rutabaga Dictyostelium ACA
n
Dictyostelium ACG Adenylyl Cyclase
Adenylyl
n
S. cerevisiae Adenylyl
Cyclase
Membrane Bound Guanylyl Cyclases
Soluble Guanylyl Cyclases
Cell 870
requires both C,, and CPa and may be regulated by their interaction. Thus, several point mutations in either C,, or C& compromise catalytic activity severely (Tang et al., 1992). Further, the amino-terminal and carboxyl-terminal halves of type I adenylyl cyclase have no catalytic activity when expressed alone, but regulated enzymatic activity is restored when they are expressed concurrently (Tang et al., 1991). Analogously, soluble forms of guanylyl cyclase are heterodimers, and each subunit contains a region that is homologous to C,, and CZa; both subunits are required for catalysis (Nakane et al., 1990). Membrane-bound guanylyl cyclases may also function as dimers or higher oligomers. The locations of the nucleotide-binding site(s) within C,, and COa are not yet known, and there are no obvious sequence homologies with other nucleotide-binding proteins. However, adenylyl cyclases are inhibited (noncompetitively with respect to Mg2+/ATP) by adenosine, 3’-AMP, and related analogs (so-called P-site inhibitors). The most obvious suggestion is that C,, and Cna represent, individually, domains for interaction with substrate and P-site inhibitors. However, the studies mentioned above suggest that both domains might contribute to the binding of both types of molecule. The apparent molecular weight of brain adenylyl cyclase (a mixture of forms) has been estimated to be 200-250 kd, based on hydrodynamic properties (Neer et al., 1980). By contrast, the actual molecular weights of the various species of adenylyl cyclase now known to be expressed in brain approximate 120 kd. Perhaps adenylyl cyclase is a dimer (actually a tetramer of Cl,- and &,-like domains). If so, catalytically inactive point mutations in C,, would be expected to complement those in C&, but they do not. It is perhaps more likely that the large apparent molecular weight of the unpurified enzyme reflects contributions from associated proteins (particularly G proteins and perhaps receptors or unidentified players). Amino acid sequence similarities among the various adenylyl cyclases are limited beyond the C,, and CZ, domains. However, as mentioned, the overall structure is well conserved from Dictyostelium to mammals-raising the question of the function of the two sets of six transmembrane spans (other than to organize or orient the two cytoplasmic structures). The extracellular domains might serve a receptor-like role, but there is little evidence for such and the amount of extracellular structure is probably small. The hydrophobic domains are presumed to be the site for activation by forskolin, a lipid-soluble diterpene. Perhaps there are endogenous counterparts in mammalian cells. It has been hypothesized that adenylyl cyclase might have a dual life, serving as a channel or transporter as well (Krupinski et al., 1989). This notion was based almost entirely on the unique topographical homology between adenylyl cyclase and various ion channels and transporters (particularly the P glycoprotein and the cystic fibrosis transmembrane conductance regulator). However, there are no sequence homologies between the adenylyl cyclases and any of the channels or transporters. Many cells secrete cyclic AMP, and Dictyostelium utilizes the cyclic
nucleotide as a “hormone” to control Cellular aggregation and differentiation. It might be reasonable to couple synthesis of cyclic AMP with its secretion by incorporating both functions into one molecule. Dictyostelium synthesizes two adenylyl cyclases-one that resembles the mammalian enzymes (AC-A) and one with a single transmembrane span (AC-G). Cyclic AMP secretion is normal when only AC-G is expressed, effectively ruling out this hypothesis (Pitt et al., 1992). Of interest, an adenylyl cyclase from Paramecium has associated pore-forming activity with selectivity for cations (observed after reconstitution into lipid bilayers) (Schultzet al., 1992). Unfortunately, the structure of this enzyme is not known. Channel activity has not been detected when mammalian adenylyl cyclases have been expressed in insect (Sf9) cells. The diversity of amino acid sequences among the hydrophobic domains of the various adenylyl cyclases would also seem to argue against their role as channels or transporters. Diverse Regulatory Properties The diverse structures of the adenylyl cyclases mirror the wide range of regulatory influences that impinge on them. At one extreme, the relatively simple soluble enzyme from Streptococcus salivarius is dependent on a-keto acids for activity. The mammalian enzymes represent the opposite pole, with type-specific stimulatory and inhibitory regulation by G protein a and by subunits, Ca*+-calmodulin, P-site inhibitors, and forskolin. One aspect of the evolution of these regulatory properties is particularly striking. The adenylyl cyclase from Saccharomyces cerevisiae is stimulated by a GTP-binding protein, as are the mammalian enzymes. Thus, this important regulatory motif is established in a primitive organism. However, the molecular players bear little relationship to one another. The stimulatory GTP-binding protein in yeast is ras, despite the fact that yeast have heterotrimeric G proteins, and the cyclase is a 230 kd peripheral membrane protein with only the slightest resemblance to the mammalian adenylyl cyclases (Kataoka et al., 1985). The properties of the eukaryotic adenylyl cyclases that have been cloned to date are presented in Table 1. All the mammalian enzymes are activated by the a subunit of G,, the G protein first identified because of this action. Some (particularly type I) are also activated by Ca2+-calmodulin. Certain enzymes are rather broadly expressed, while others are very restricted in their distribution (particularly type Ill) (Sakalyar and Reed, 1990). One cloned adenylyl cyclase from Drosophila (the product of the rutabaga gene) is similar biochemically to the mammalian type I enzyme. However, the molecule is remarkable in that it has a very long (110 kd) carboxyl-terminal extension of unknown significance. A missense mutation in the rutabaga adenylyl cyclase destroys catalytic activity and is associated with a learning deficiency in the fly (Levin et al., 1992). The adenylyl cyclases (from murine S49 cells and platelets) that were originally characterized with regard to the effects of G proteins are activated by G,, and are largely insensitive to the G protein by subunit complex. Similar results with other G protein-regulated effecters (particularly retinal cyclic GMP phosphodiesterase) led to the concept that G protein a subunits regulate effecters, while pr
Minireview 071
Table
1. Properties
of Eukaryotic
Adenylyl
Cyclases Effect
Type” I II III IV V VI rutabaga AC-A AC-G CYRl
Amino
Acid
Residues
1134 1090 1144 1964 1184 1165 2249 1407 658
of G Proteins
Expression
Pr
Brain Brain, lung Olfactory Brain, others Heart, brain, others Heart, brain, others Mushroom body During aggregation Fruiting body Constitutive
+ 0 + 0 0 0 ? ?d 0
Ca2+-Calmodulin
Forskolin
+ 0 + 0 0 0 + ? ?d 0
+ + + t t + + 0 0 0
B Adenylyl cyclase types I-VI are mammalian; rutabaga is from Drosophila; AC-A and AC-G are from Dictyostelium; CYRI is from Saccharomyces cerevisiae. References: I (Krupinski et al., 1989) II (Feinstein et al., 1991) Ill (Sakalyar and Reed, 1990) IV (Gao and Gilman, 1991) V (Ishikawa et al., 1992) VI (Premont et al., 1992; Katsushika et al., 1992) rutabaga (Levin et al., 1992) AC-A and AC-G (Pitt et al., 1992) and CYRl (Kataoka et al., 1985). b +, stimulates; -, inhibits; 0, no effect. c AC-A is activated by GTPTS; however, a homolog of G,a has not been detected in Dictyostelium. d Unlikely to have an effect; GTPyS does not activate. n Activated bv ras.
was assigned less interesting roles. Therefore, when the novel adenylyl cyclase clones were expressed and examined individually, it was surprising to detect prominent, type-specific regulation of enzymatic activity by by. Whereas 9~ inhibits type I adenylyl cyclase when stimulated by either G., or Ca2+-calmodulin, fir greatly potentiates the stimulatory effect of G,, on either type II or type IV adenylyl cyclase (Tang and Gilman, 1991). The other adenylyl cyclases cloned to date are relatively unaffected by Pr. The concentration of by required to modulate adenylyl cyclase activity is significantly greater than that of G,a, and thus it appears that the G. oligomer would not be the source of 9~ for such regulation. (Besides, it is unclear what the point would be of simultaneously stimulating and
inhibiting type I adenylyl cyclase by dissociation of the activated subunits of G,). Rather, it is assumed that the necessary concentrations of S+r are contributed by G proteins such as Gi and G,, which are far more abundant than G, (at least in brain). This provides a clear mechanism for cross-talk and interaction between signaling pathways. Adenylyl cyclase is activated by G,-linked pathways but can be inhibited or stimulated further by 9~ when other G protein-mediated pathways are activated simultaneously (Figure 2). Such conditional, synergistic activation of type II adenylyl cyclase by G,- and Gi-mediated pathways has been demonstrated in intact cells by transient expression of cDNAsencoding the requisite proteins(Federman et al., 1992). This mechanism may well underlie the synergistic effects of neurotransmitters on cyclic AMP accumulation
Kinase (?) Figure
2. Regulation
of Mammalian
Adenylyl
Cyclases
See text for description. R. G protein-coupled receptors; G protein a subunit; CaM, calmodulin; 3’-AMP, a P-site
G, and G,, G proteins inhibitor.
that stimulate
and inhibit adenylyl
cyclase,
respectively;
Ga, any
Cdl 872
in brain slices, which were documented 20 years ago (Sattin et al., 1975). Regulation of effecters by G protein 6~ subumts may well be a general phenomenon. Although the effects of 67 on K+ channels remain controversial, 67 has recently been shown to activate one form of phospholipase C from HL60 cells (Camps et al., 1992). The question of specificity then arises, since four isoforms of 9 and an increasing number of distinctrsubunits have been identified. Defined 6~ complexes can be assembled by expression in Sf9 cells and purified facilely by subunit affinity chromatography. There is some specificity in their interactions with adenylyl cyclase, and this interaction is dependent on prenylation of the G protein y subunit (IAiguez-Lluhi et al., 1992). Receptors that interact with members of the Gi family of G proteins (Gil, G,2, and Gis) mediate inhibition of adenylyl cyclase. Although the G, proteins are demonstrably important (revealed by reconstitution of membranes with Gi oligomers), mechanisms are uncertain and may vary with the type of cyclase in question. Transfection of cells with constitutively active mutant forms of G,a results in inhibition of cyclic AMP accumulation, implicating a subunits in the pathway (Wong et al., 1992). However, activated Gia subunits are very weak inhibitors (at best) of adenylyl cyclase when tested in vitro-in membranes or with purified cyclases. Perhaps Gia proteins inhibit adenyfyl cyclase indirectly by a pathway that remains to be reconstituted in vitro. However, when G protein 6~ subunits are tested in vitro. inhibitory effects are observed readily. This effect is y on type I adenylyl cyclase. The effect is her cyclases and is apparently due to deactiry f3r (Gilman, 1964). Although the effort that understanding mechanisms of inhibition of adenylyl cyclase by the G, proteins may exceed the physiological significance of the phenomenon, the solution of this problem will almost certainly yield valuable insights into the scope and mechanism of action of this important group of G proteins. References Bakalyar, H. A. and Reed, R. R. (1990). Science 250, 1403-1406 Camps, M., Hou, C. F., Sidiropoulos, D., Stock, J. B., Jakobs, K. H., and Gierschik, P. (1992). Eur. J. Biochem. 206, 821-831. Chinkers. M. and Garbers, D. L. (1991). Annu. Rev. Biochem. 60,553575. Federman, A. D., Conklin, B. R., Schrader, Bourne, H. R. (1992). Nature 356, 159-161.
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