Cyclic AMP-induced conformational changes in mycobacterial protein acetyltransferases

The activities of a number of proteins are regulated by the binding of cAMP and cGMP to cyclic nucleotide binding (CNB) domains that are found associated with one or more effector domains with diverse functions. Although the conserved architecture of CNB domains has been extensively studied by x-ray crystallography, the key to unraveling the mechanisms of cAMP action has been protein dynamics analyses. Recently, we have identified a novel cAMP-binding protein from mycobacteria, where cAMP regulates the activity of an associated protein acetyltransferase domain. In the current study, we have monitored the conformational changes that occur upon cAMP binding to the CNB domain in these proteins, using a combination of bioluminescence resonance energy transfer and amide hydrogen/deuterium exchange mass spectrometry. Coupled with mutational analyses, our studies reveal the critical role of the linker region (positioned between the CNB domain and the acetyltransferase domain) in allosteric coupling of cAMP binding to activation of acetyltransferase catalysis. Importantly, major differences in conformational change upon cAMP binding were accompanied by stabilization of the CNB and linker domain alone. This is in contrast to other cAMP-binding proteins, where cyclic nucleotide binding has been shown to involve intricate and parallel allosteric relays. Finally, this powerful convergence of results from bioluminescence resonance energy transfer and hydrogen/deuterium exchange mass spectrometry reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins in the absence of high resolution structural information.     

The cyclic nucleotide specificity of eight cAMP-binding proteins in Dictyostelium discoideum is correlated into three groups

cAMP is a mediator of inter- and intracellular events in Dictyostelium discoideum and is thought to act through specific receptors. Eight forms of cAMP-binding proteins have been described in this organism: four forms of a cell surface receptor, a cell surface and extracellular phosphodiesterase, an intracellular cAMP-dependent protein kinase (CAK), and a recently identified cAMP-binding protein (CABP1) that is present on the cell surface, in the cytoplasm, and in the nucleus. In this study we have analyzed the cyclic nucleotide specificity of these cAMP-binding proteins using 13 derivatives of cAMP with modifications in the adenine, ribose, and phosphate moiety. The results suggest that the cAMP-binding proteins belong to three groups: (i) four forms of the cell surface receptor, (ii) two forms of an intracellular receptor (CABP1 and CAK), and (iii) cell surface and extracellular phosphodiesterase. cAMP is probably bound to the surface receptors in the anti conformation in a hydrophobic cleft of the receptor with essential interactions at N6H2' and O3'. In contrast, cAMP is probably bound to CAK and CABP1 in the syn conformation with essential interactions at O2', O3', O5', and exocyclic oxygen. Finally, binding of cAMP to phosphodiesterase involves only O3' and exocyclic oxygen. The cyclic nucleotide specificity of cAMP-induced processes in D. discoideum indicates that the cell surface receptors participate in the transduction of the cAMP signal during chemotaxis and cell differentiation. Functions for CABP1 and CAK in these processes are presently elusive.     

cAMP-regulated Protein Lysine Acetylases in Mycobacteria

Cyclic AMP synthesized by Mycobacterium tuberculosis has been shown to play a role in pathogenesis. However, the high levels of intracellular cAMP found in both pathogenic and non-pathogenic mycobacteria suggest that additional and important biological processes are regulated by cAMP in these organisms. We describe here the biochemical characterization of novel cAMP-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) that contain a cyclic nucleotide binding domain fused to a domain that shows similarity to the GNAT family of acetyltransferases. We detect protein lysine acetylation in mycobacteria and identify a universal stress protein (USP) as a substrate of MSMEG_5458. Acetylation of a lysine residue in USP is regulated by cAMP, and using a strain deleted for MSMEG_5458, we show that USP is indeed an in vivo substrate for MSMEG_5458. The Rv0998 protein shows a strict cAMP-dependent acetylation of USP, despite a lower affinity for cAMP than MSMEG_5458. Thus, this report not only represents the first demonstration of protein lysine acetylation in mycobacteria but also describes a unique functional interplay between a cyclic nucleotide binding domain and a protein acetyltransferase.     

Dissecting interdomain communication within cAPK regulatory subunit type IIbeta using enhanced amide hydrogen/deuterium exchange mass spectrometry (DXMS)

cAMP-dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dimer bound to two catalytic (C) subunits and is involved in numerous cell signaling pathways. The C-subunit is activated allosterically when two cAMP molecules bind sequentially to the cAMP-binding domains, designated A and B (cAB-A and cAB-B, respectively). Each cAMP-binding domain contains a conserved Arg residue that is critical for high-affinity cAMP binding. Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP-binding domains, and cAPK activation. To better understand the local and long-range effects that the Arg-to-Lys mutation has on the dynamic properties of the R-subunit, the amide hydrogen/deuterium exchange in the RIIbeta subunit was probed by electrospray mass spectrometry. Mutant proteins containing the Arg-to-Lys substitution in either cAMP-binding domain were deuterated for various times and then, prior to mass spectrometry analysis, subjected to pepsin digestion to localize the deuterium incorporation. Mutation of this Arg in cAB-A (Arg230) causes an increase in amide hydrogen exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is indicative of the importance of this Arg in domain organization. Mutation of Arg359 (cAB-B) leads to increased exchange in the adjacent cAB-A domain, particularly in the cAB-A domain C-helix that lies on top of the cAB-B domain and is believed to be functionally linked to the cAB-B domain. This interdomain communication appears to be a unidirectional pathway, as mutation of Arg230 in cAB-A does not effect dynamics of the cAB-B domain.     

Novel single chain cAMP sensors for receptor-induced signal propagation

cAMP is a universal second messenger of many G-protein-coupled receptors and regulates a wide variety of cellular events. cAMP exerts its effects via cAMP-dependent protein kinase (PKA), cAMP-gated ion channels, and two isoforms of exchange protein directly activated by cAMP (Epac). Here we report the development of novel fluorescent indicators for cAMP based on the cAMP-binding domains of Epac and PKA. Fluorescence resonance energy transfer between variants of green fluorescent protein (enhanced cyan fluorescent protein and enhanced yellow fluorescent protein) fused directly to the cAMP-binding domains was used to analyze spatial and temporal aspects of cAMP-signaling in different cells. In contrast to previously developed PKA-based indicators, these probes are comprised of only a single binding site lacking cooperativity, catalytic properties, and interactions with other proteins and thereby allow us to easily image free intracellular cAMP and rapid signaling events. Rapid beta-adrenergic receptor-induced cAMP signals were observed to travel with high speed ( approximately 40 microm/s) throughout the entire cell body of hippocampal neurons and peritoneal macrophages. The developed indicators could be ubiquitously applied to studying cAMP, its physiological role and spatio-temporal regulation.     

PKA-I holoenzyme structure reveals a mechanism for cAMP-dependent activation

Protein kinase A (PKA) holoenzyme is one of the major receptors for cyclic adenosine monophosphate (cAMP), where an extracellular stimulus is translated into a signaling response. We report here the structure of a complex between the PKA catalytic subunit and a mutant RI regulatory subunit, RIalpha(91-379:R333K), containing both cAMP-binding domains. Upon binding to the catalytic subunit, RI undergoes a dramatic conformational change in which the two cAMP-binding domains uncouple and wrap around the large lobe of the catalytic subunit. This large conformational reorganization reveals the concerted mechanism required to bind and inhibit the catalytic subunit. The structure also reveals a holoenzyme-specific salt bridge between two conserved residues, Glu261 and Arg366, that tethers the two adenine capping residues far from their cAMP-binding sites. Mutagenesis of these residues demonstrates their importance for PKA activation. Our structural insights, combined with the mutagenesis results, provide a molecular mechanism for the ordered and cooperative activation of PKA by cAMP.     

cAMP-dependent protein kinase regulatory subunit type IIbeta: active site mutations define an isoform-specific network for allosteric signaling by cAMP

cAMP-dependent protein kinase (cAPK) contains a regulatory (R) subunit dimer bound to two catalytic (C) subunits. Each R monomer contains two cAMP-binding domains, designated A and B. The sequential binding of two cAMPs releases active C. We describe here the properties of RIIbeta and two mutant RIIbeta subunits, engineered by converting a conserved Arg to Lys in each cAMP-binding domain thereby yielding a protein that contains one intact, high affinity cAMP-binding site and one defective site. Structure and function were characterized by circular dichroism, steady-state fluorescence, surface plasmon resonance and holoenzyme activation assays. The Ka for RIIbeta is 610 nM, which is 10-fold greater than its Kd(cAMP) and significantly higher than for RIalpha and RIIalpha. The Arg mutant proteins demonstrate that the conserved Arg is important for both cAMP binding and organization of each domain and that binding to domain A is required for activation. The Ka of the A domain mutant protein is 21-fold greater than that of wild-type and the Kd(cAMP) is increased 7-fold, confirming that cAMP must bind to the mutated site to initiate activation. The domain B mutant Ka is 2-fold less than its Kd(cAMP), demonstrating that, unlike RIalpha, cAMP can access the A site even when the B site is empty. Removal of the B domain yields a Ka identical to the Kd(cAMP) of full-length RIIbeta, indicating that the B domain inhibits holoenzyme activation for RIIbeta. In RIalpha, removal of the B domain generates a protein that is more difficult to activate than the wild-type protein.     

DNA binding by cyclic adenosine 3',5'-monophosphate dependent protein kinase from calf thymus nuclei.

Cyclic adenosine 3',5'-monophosphate (cAMP) dependent protein kinase and proteins specifically binding cAMP have been extracted from calf thymus nuclei and analyzed for their abilities to bind to DNA. Approximately 70% of the cAMP-binding activity in the nucleus can be ascribed to a nuclear acidic protein with physical and biochemical characteristics of the regulatory (R) subunit of cAMP-dependent protein kinase. Several peaks of protein kinase activity and of cAMP-binding activity are resolved by affinity chromatography of nuclear acidic proteins on calf thymus DNA covalently linked to aminoethyl Sephrarose 4B. When an extensively purified protein kinase is subjected to chromatography on the DNA column in the presence of 10(-7) M cAMP, the R subunit of the kinase is eluted from the column at 0.05 M NaCl while the catalytic (C) subunit of the enzyme is eluted at 0.1-0.2 M NaCl. When chromatographed in the presence of histones, the R subunit is retained on the column and is eluted at 0.6-0.9 M NaCl. In the presence of cAMP, association of the C subunit with DNA is enhanced, as determined by sucrose density gradient centrifugation of DNA-protein kinase complexes. cAMP increases the capacity of the calf thymus cAMP-dependent protein kinase preparation to bind labeled calf thymus DNA, as determined by a technique employing filter retention of DNA-protein complexes. This protein kinase preparation binds calf thymus DNA in preference to salmon DNA, Escherichia coli DNA, or yeast RNA. Binding of protein kinases to DNA may be part of a mechanism for localizing cyclic nucleotide stimulated protein phosphorylation at specific sites in the chromatin.     

Activation of type I cyclic AMP-dependent protein kinases with defective cyclic AMP-binding sites

Two S49 mouse lymphoma cell variants hemizygous for expression of mutant regulatory (R) subunits of type I cyclic AMP-dependent protein kinase were used to investigate functional consequences of lesions in the putative cAMP-binding sites of R subunit. Kinase activation properties of wild-type and mutant enzymes were compared using cAMP and six site-selective analogs of cAMP. Kinases from both mutant sublines were relatively resistant to cyclic nucleotide-dependent activation, but they were fully activable by at least some effectors. Relative resistances of the mutant kinases varied from about 5-fold for analogs selective for their nonmutated sites to as much as 700-fold for analogs selective for their mutated sites; resistance to cAMP was intermediate. Apparent affinities of wild-type and mutant R subunits for [3H]cAMP were not appreciably different, but competition experiments with site-selective analogs of cAMP suggested that binding of cAMP to mutant R subunits was primarily to their nonmutated sites. Analyses of cooperativity in cyclic nucleotide-dependent activation of mutant kinases, synergism between site I- and site II-selective analogs in activating the mutant enzymes, and dissociation of bound cAMP from mutant R subunits provided additional evidence that the mutations in these strains selectively inactivated single classes of cAMP-binding sites: phenomena attributable in wild-type enzyme to intrachain interactions between sites I and II were always absent or severely diminished in experiments with the mutant enzymes. These results confirm that R subunit sequences implicated in cAMP binding by homology with other cyclic nucleotide-binding proteins actually correspond to functional cAMP-binding sites. Furthermore, occupation of either cAMP-binding site I or II is apparently sufficient for activation of cAMP-dependent protein kinase. The presence of four functional cAMP-binding sites in wild-type kinase enhances the cooperativity and sensitivity of cAMP-mediated activation.     

“cAMP Sponge”: A Buffer for Cyclic Adenosine 3′, 5′-Monophosphate

Background

While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP).

Methods/Principal Findings

Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIβ of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named “cAMP sponge”) was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets.

Conclusions

This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.     

Rearrangements in a hydrophobic core region mediate cAMP action in the regulatory subunit of PKA

cAMP-dependent protein kinase (PKA) forms an inactive heterotetramer of two regulatory (R; with two cAMP-binding domains A and B each) and two catalytic (C) subunits. Upon the binding of four cAMP molecules to the R dimer, the monomeric C subunits dissociate. Based on sequence analysis of cyclic nucleotide-binding domains in prokaryotes and eukaryotes and on crystal structures of cAMP-bound R subunit and cyclic nucleotide-free Epac (exchange protein directly activated by cAMP), four amino acids were identified (Leu203, Tyr229, Arg239 and Arg241) and probed for cAMP binding to the R subunits and for R/C interaction. Arg239 and Arg241 (mutated to Ala and Glu) displayed no differences in the parameters investigated. In contrast, Leu203 (mutated to Ala and Trp) and Tyr229 (mutated to Ala and Thr) exhibited up to 30-fold reduced binding affinity for the C subunit and up to 120-fold reduced binding affinity for cAMP. Tyr229Asp showed the most severe effects, with 350-fold reduced affinity for cAMP and no detectable binding to the C subunit. Based on these results and structural data in the cAMP-binding domain, a switch mechanism via a hydrophobic core region is postulated that is comparable to an activation model proposed for Epac.     

Purification and characterization of two high-affinity (adenosine 3',5'-monophosphate)-binding proteins from yeast. Identification as multiple forms of glyceraldehyde-3-phosphate dehydrogenase

Two high-affinity cAMP-binding proteins (I and II) have been purified to homogeneity from baker's yeast by a procedure avoiding proteolytic damage. These proteins have been identified as multiple forms of glyceraldehyde-3-phosphate dehydrogenase. The two cAMP-binding proteins are similar in affinities for cAMP, have identical elution volumes on gel filtration, and contain one type of subunit (Mr 37 500). The form II of glyceraldehyde-3-phosphate dehydrogenase is free of NAD+ and has a Kd of 1.3 X 10(-6) M with respect to cAMP. A marked concentration-dependent self-association of the subunits of the form-II protein was revealed by Yphantis sedimentation equilibrium studies. Significant monomer concentrations are present at total concentrations less than 0.02 mg/ml. Conventional sedimentation equilibrium analyses indicated a tetramer Mr of 170 000. The high-affinity binding of cAMP to glyceraldehyde-3-phosphate dehydrogenase may significantly reduce intracellular cAMP levels and is also discussed in relation to the nature of eukaryote cAMP-binding proteins with similar native or subunit Mr values which are at present functionally undefined.     

Purification and characterization of a membrane-associated cAMP-binding protein from developing Dictyostelium discoideum

Plasma membranes of 6-h differentiated Dictyostelium discoideum cells contain a cAMP-binding protein with the properties ascribed to the chemotaxis receptor present on these cells. We have purified this cAMP-binding protein using DEAE-Sephadex chromatography, hydrophobic chromatography on decylagarose and preparative polyacrylamide gel electrophoresis in nonionic detergent. Photoaffinity labeling of the DEAE-purified material with 8-azido-[32P] cAMP shows that only an Mr = 70,000 species on sodium dodecyl sulfate gels contains a cAMP-binding site. Two-dimensional polyacrylamide gel electrophoresis of material eluted from decyl-agarose and photoaffinity labeled indicates that the cAMP-binding protein is the most acidic of many Mr = 70,000 proteins present. This method is readily scaled up to process up to 10(11) cells which yield from 25 to 100 micrograms of cAMP-binding protein. Nucleotide specificity studies established that the cAMP-binding site of the protein is similar to that of the cAMP receptor assayed on intact cells and membranes. The rates of association and dissociation of the cAMP-binding protein are extremely rapid as found for the receptor, and its affinity for cAMP is comparable. The cAMP-binding protein is a concanavalin A binding glycoprotein, and is resistant to proteolysis by trypsin, but not chymotrypsin. Like the cAMP receptor in membranes and crude detergent extracts, this cAMP-binding protein is inhibited by phenylmethylsulfonyl fluoride. The purified binding protein exists in solution largely as a monomeric species, with some dimer being detected on gel filtration. Based on these criteria, we conclude that this cAMP binding protein represents the binding subunit of the cAMP chemotaxis receptor.     

Alteration in cyclic adenosine 3':5'-monophosphate binding proteins during the phenotypic change of mouse macrophages transformed by a temperature-sensitive mutant (tsA640) of SV40

By using a photoaffinity ligand, cell extracts from transformed macrophages that were established by infection with temperature-sensitive mutants (tsA640) of simian virus 40 (SV40) were examined for cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins. At the nonpermissive temperature for SV40 large T antigen, 39.0 degrees C, no significant cAMP-binding proteins could be detected, such as primary mouse macrophages. At the permissive temperature of 33.0 degrees C, cAMP-binding proteins appeared later than SV40 T antigen expression and cellular DNA synthesis. The profile of cAMP-binding proteins was similar to that of resting, but not proliferating, mouse clonal fibroblasts (BALB/c 3T3). These and previous results suggest that SV40 T antigen influences the expression of cAMP-binding proteins in tsA640-transformed macrophages; the large/small T antigen converts the profile of cAMP-binding proteins from macrophage to fibroblastic cells.     

Two lipid-anchored cAMP-binding proteins in the yeast Saccharomyces cerevisiae are unrelated to the R subunit of cytoplasmic protein kinase A.

We show that the yeast, Saccharomyces cerevisiae, contains two cAMP-binding proteins in addition to the well-characterized regulatory (R) subunit of cytoplasmic cAMP-dependent protein kinase (PKA). We provide evidence that they comprise a new type of cAMP receptor, membrane-anchored by covalently attached lipid structures. They are genetically not related to the cytoplasmic R subunit. The respective proteins can be detected in sral mutants, in which the gene for the R subunit of PKA has been disrupted and a monoclonal antibody raised against the cytoplasmic R subunit does not cross-react with the two membrane-bound cAMP-binding proteins. In addition, they differ from the cytoplasmic species also with respect to their location and the peptide maps of the photoaffinity-labeled proteins. Although they differ from one another in molecular mass and subcellular location, peptide maps of the cAMP-binding domains resemble each other and both proteins are membrane-anchored by lipid structures, one to the outer surface of the plasma membrane, the other to the outer surface of the inner mitochondrial membrane. Both anchors can be metabolically labeled by Etn, myo-Ins and fatty acids. In addition, the anchor structure of the cAMP receptor from plasma membranes can be radiolabeled by GlcN and Man. After cleavage of the anchor with glycosylphosphatidylinositol-specific phospholipase C from trypanosomes, the solubilized cAMP-binding protein from plasma membranes reacts with antibodies which specifically recognize the cross-reacting determinant from soluble trypanosomal coat protein, suggesting similarity of the anchors. Degradation studies also point to the glycosylphosphatidylinositol nature of the anchor from the plasma membrane, whereas the mitochondrial counterpart is less complex in that it lacks carbohydrates. The plasma membrane cAMP receptor is, in addition, modified by an N-glycosidically linked carbohydrate side chain, responsible mainly for its higher molecular mass.     

A simple electrostatic switch important in the activation of type I protein kinase A by cyclic AMP

Cyclic AMP activates protein kinase A by binding to an inhibitory regulatory (R) subunit and releasing inhibition of the catalytic (C) subunit. Even though crystal structures of regulatory and catalytic subunits have been solved, the precise molecular mechanism by which cyclic AMP activates the kinase remains unknown. The dynamic properties of the cAMP binding domain in the absence of cAMP or C-subunit are also unknown. Here we report molecular-dynamics simulations and mutational studies of the RIalpha R-subunit that identify the C-helix as a highly dynamic switch which relays cAMP binding to the helical C-subunit binding regions. Furthermore, we identify an important salt bridge which links cAMP binding directly to the C-helix that is necessary for normal activation. Additional mutations show that a hydrophobic "hinge" region is not as critical for the cross-talk in PKA as it is in the homologous EPAC protein, illustrating how cAMP can control diverse functions using the evolutionarily conserved cAMP-binding domains.     

Epac: a new cAMP target and new avenues in cAMP research

Five years ago, Epac--a guanine nucleotide exchange factor for the Ras-like small GTPases Rap1 and Rap2--was found to be a new target of cyclic AMP, which opened up new avenues for cAMP research. Structural analysis of the cAMP-binding domains of Epac2 has identified a unifying mechanism for how cAMP activates proteins, and the design and synthesis of an Epac-specific cAMP analogue has paved the way for future discoveries.     

Comparison of adenylate cyclase and cAMP-dependent protein kinase in gametocytogenic and nongametocytogenic clones of Plasmodium falciparum

Adenylate cyclase and cAMP-dependent protein kinase activities in gametocytogenic (LE5) and nongametocytogenic (T9/96) clones of Plasmodium falciparum were compared to explore the role of cAMP in sexual differentiation of the parasite. Basal adenylate cyclase levels were equivalent in the 2 clones. However, cAMP-dependent histone II-A kinase activity was significantly higher in LE5 than in T9/96 over a range of cAMP concentrations. This difference was due to a decreased Vmax for the enzyme in the nongametocytogenic clone and not to an increased Ka for cAMP. Examination of parasite cAMP-binding proteins, likely to be kinase regulatory subunits, by both photoaffinity labeling with [32P]8-N3-cAMP and affinity chromatography of metabolically [35S]methionine-labeled cytosol of cAMP-agarose revealed a 53-kDa cAMP binding protein in both clones and a 49-kDa cAMP-binding protein in T9/96 that was absent in LE5. Our results suggest that T9/96 has lost the ability to undergo gametocytogenesis due to a substantial decrease in cAMP-dependent protein kinase activity rendering the parasite unable to respond to increased intracellular cAMP levels. Moreover, the reduction in cAMP-dependent protein kinase activity may be due to the presence of an alternative regulatory subunit of the kinase.     

Neurohormonal receptors and cyclic AMP-binding proteins in rabbit tracheal mucosa-submucosa

Neurohormones and drugs that alter in vitro tracheal electrolyte transport and mucus glycoprotein secretion were examined for their ability to alter cyclic nucleotide accumulation in a smooth muscle-free preparation of rabbit tracheal mucosa-submucosa. cAMP levels were increased by beta-adrenergic agonists, histamine, 2-Cl-adenosine and prostaglandin E1. cGMP levels were increased by carbachol. The phosphodiesterase inhibitor isobutylmethylxanthine increased cAMP and cGMP levels and potentiated only the beta-adrenergic effects. The beta-adrenergic effects were blocked by (+/-)-propranolol and the effects of histamine by diphenhydramine, atropine and (+/-)-propranolol. Atropine blocked the carbachol effects. The isolated surface epithelium from rabbit trachea had higher basal cAMP levels and greater response to beta-adrenergic agonists and isobutylmethylxanthine than the mucosa-submucosa. Two major cAMP-binding proteins in the tracheal mucosa-submucosa were identified with the photoaffinity label 8-N3-[32P]cAMP. Agents that increased cAMP levels also decreased photoaffinity labelling, suggesting that these two cAMP-binding proteins were being occupied in the intact cell. The molecular weights of the proteins were 50 000 and 54 000 and correspond in electrophoretic mobility to the regulatory subunits of Type-I and Type-II cAMP-dependent protein kinases, respectively. The results are consistent with the hypothesis that epithelial functions in the airways are modulated by a number of agonists which increase cyclic nucleotide levels. The effects of beta-adrenergic agonists is apparently mediated by activation of adenylate cyclase and subsequent activation of cAMP-dependent protein kinases.