cAMP (Cyclic Adenosine 3,5-monophosphate) is the first identified second messenger, which has a fundamental role in the cellular response to many extracellular stimuli. The cAMP signaling pathway controls a diverse range of cellular processes. Indeed, not only did cAMP provide the paradigm for the second messenger concept, but also provided the paradigm for signaling compartmentalization. The different receptors, chiefly the GPCRs (G-Protein Coupled Receptors), Alpha/Beta-ADRs (Adrenergic Receptors), Growth Factor receptors, CRHR (Corticotropin Releasing Hormone Receptor), GcgR (Glucagon Receptor), DCC (Deleted in Colorectal Carcinoma), etc are responsible for cAMP accumulation in cells that cause different physiological outcomes, and changes in cAMP levels effects the selective activation of PKA(cAMP dependent Protein Kinase-A) isoforms. The chief source of cAMP is from ATP (Adenosine Triphosphate). In mammals, the conversion of ATP to cAMP is mediated by members of the Class-III AC (Adenylyl Cyclase)/ADCY (Adenylate Cyclase) family (E.C 126.96.36.199), which in humans comprises nine trans-membrane AC enzymes or tmACs
and one soluble AC or sAC (Ref.1 & 2). The sAC predominantly occurs in mature spermatozoa. tmACs are regulated by heterotrimeric G-proteins in response to the stimulation of various types of GPCRs and therefore play a key role in the cellular response to extracellular signals. sAC, in contrast, is insensitive to G-proteins. Instead, sAC is directly activated by Ca2+ (Calcium Ions) and the metabolite HCO3- (Bicarbonate Ions), rendering the enzyme an intracellular metabolic sensor. Together, tmACs and sAC regulate a diverse set of essential biological processes, such as differentiation and gene transcription, and this makes cAMP signaling, an important mediator of intra- and extracellular signals in organisms from prokaryotes to higher eukaryotes (Ref.2 & 3).
The extracellular stimuli like neurotransmitters, hormones, inflammatory stimuli, stress, epinephrine, norepinephrine, etc activate the G-proteins through receptors like GPCRs and ADR-Alpha/Beta. The major G-proteins that regulate activation of ACs are the GN-AlphaS (GN-AlphaS Complex Locus), GN-AlphaQ (Guanine Nucleotide-Binding Protein-Alpha-Q) and GN-AlphaI (Guanine Nucleotide Binding Protein-Alpha Inhibiting Activity Polypeptide). Upon activation these subunits are separated from GN-Beta (Guanine Nucleotide-Binding Protein-Beta) and GN-Gamma (Guanine Nucleotide-Binding Protein-Gamma) subunits and are converted to their GTP bound states that exhibit distinctive regulatory features on the nine tmACs in order to regulate intracellular cAMP levels (Ref.1 & 4). Other ligands like Gcg (Glucagon), Ucns (Urocortins) and Ntn1 (Netrin-1), etc either directly regulate activity of ACs or via G-protein activation through their respective receptors like GcgR, CRHR and DCC. GN-AlphaS and GN-AlphaQ activate ACs to increase intracellular cAMP levels, where as, GN-AlphaI decrease intracellular cAMP levels by inhibiting ACs. GN-Beta and GN-Gamma subunits act synergistically with GN-AlphaS and GN-AlphaQ only to activate ACII, ACIV and ACVII. However the Beta and Gamma-subunits along with GN-AlphaI inhibit the activity of ACI, ACV and ACVI. ACI, ACIII, ACV, ACVI and ACIX isoforms play a vital role in spinal pain transmission and are up-regulated by chronic Opoids, for which they are often inhibited by Ca2+ and other proteins acting downstream to cAMP signaling. GN-AlphaI activity is counteracted by RGS (Regulator of G-Protein Signaling) proteins (Ref.5, 6 & 7). However, Beta-Arrestins desensitize many GPCRs and ADRs and this blocks interaction between receptors and their cellular effectors, thereby inhibiting GPCR activity. Beta-Arrestins contol intracellular cAMP levels by switching of signaling from GN-AlphaS to GN-AlphaI by recruiting PDE4D (cAMP Specific Phosphodiesterase-4D) isoforms (Ref.8). Growth factors and PI3K (Phosphatidylinositde-3 Kinase) crosstalk with cAMP signaling by activating Akt (v-Akt Murine Thymoma Viral Oncogene Homolog), which further activates PDE (Phosphodiesterase) to facilitate the conversion of cAMP to AMP through Akt Signaling. This modulates cardiac contractility and release of metabolic energy. G-proteins indirectly influence cAMP signaling by activating PI3K and PLC (Phospholipase-C). PLC cleaves PIP2 (Phosphatidylinositol 4,5-bisphosphate) to generate DAG (Diacylglycerol) and IP3 (Inositol 1,4,5-trisphosphate). DAG in turn activates PKC (Protein Kinase-C). IP3 modulates proteins upstream to cAMP signaling with the release of Ca2+ through IP3R (IP3 Receptor) facilitating activation of Src (v-Src Avian Sacroma (Schmidt-Ruppin A-2)Viral Oncogene) along with PYK2 (Proline-Rich Tyrosine Kinase-2). Activation of Src by PI3K and growth factors enhance activation of Raf1 (v-Raf1 Murine Leukemia Viral Oncogene Homolog-1) through Ras. Raf1 facilitate MEK1 (MAPK/ERK Kinase-1) and MEK2 activation, which in turn activates ERKs (Extracellular Signal-Regulated Kinases) and this ultimately leads to induction of transcription regulator Elk1 (ETS-domain protein Elk1) mediated gene expression. PKC modulate cAMP signaling by activation of Raf1, PYK2 and ACs like ACI, ACII, ACIII, ACV and ACVII, but inhibits ACVI (Ref.9).
Once active, the tmACs and sAC produces the second messenger cAMP in response to a wide range of signal transduction pathways. Three main targets of cAMP are PKA, the GTP-exchange protein, EPACs (Exchange Protein Activated by cAMP) and the CNG (Cyclic-Nucleotide Gated Ion Channel). CNG activation by cAMP provides passage to Ca2+ influx. cAMP activate Rap1A (Ras-Related Protein-1A) and Rap1B (Ras-Related Protein Rap1B) through the PKA-independent and EPAC (Exchange Protein Activated by cAMP-dependent pathway. cAMP activates cAMP-GEFI (cAMP-Regulated Guanine Nucleotide Exchange Factor-I)/EPAC1 and cAMP-GEFII (cAMP-Regulated Guanine Nucleotide Exchange Factor-II)/EPAC2 that in turn activate Rap1A and Rap1B, respectively. Rap1A and Rap1B then forms an active complex with BRaf (v-Raf Murine Sarcoma Viral Oncogene Homolog-B1) for MEK1/2 activation finally resulting in Elk1 activation. Rap1A and Rap1B further stimulate Rap1 and Rap2 pathways that are vital for cell survival (Ref.10 & 11). Apart from CNG, PKC, and EPACs, other direct targets of cAMP includes, PDE, mTOR (Mammalian Target of Rapamycin), p70S6K/RPS6KB1 (Ribosomal Protein-S6 kinase-70kDa-Polypeptide-1), PLA2 (Phospholipase-A2). cAMP-activated mTOR and p70S6K promote cell growth via the mTOR and p70S6K signaling route, whereas, PLA2 facilitates release of stored energy by enhancing the Fatty acid metabolism processes. The Urocortin-cAMP mediated induction of PKC and p38 results in Apoptosis and Cytokine production (like that of IL-6 (Interleukin-6)), downstream to the Urocortin-cAMP pathway. Although cAMP directly regulates the activities of some molecules, PKA appears to be the major ‘read-out’ for cAMP and is the predominant cellular effector of cAMP (Ref.12). PKA is tethered to specific cellular locations by a growing class of proteins called AKAPs (A-Kinase Anchor Proteins). Targeting of PKA isozymes by AKAPs is important for an increasing number of physiological processes such as cAMP regulation of ion channels in the nervous system, regulation of the cell cycle which involves microtubule dynamics, chromatin condensation and decondensation, nuclear envelope disassembly and reassembly, Steroidogenesis, reproductive function, immune responses and numerous intracellular transport mechanisms (Ref.13).
In the following sections, the role of localized pools of PKA in the context of some selected physiological processes where regulation by cAMP plays a major role has been analyzed. The ADR-Alpha/Beta stimulation of cAMP-PKA phosphorylates several proteins related to excitation-contraction coupling like activation of L-Type CaCn (Calcium Channels), KCn (Potassium Channel), SCn
(Sodium Channels), ClCn (Chloride Channels), RyR (Ryanodine Receptor), Pln (Phospholamban), CRP (C-Reactive Protein) but inhibts TnnI (Troponin-I). PKA phosphorylates Pln that regulates the activity of SERCA2 (Sarcoplasmic Reticulum Ca2+-ATPase-2). It leads to increased reuptake of Ca2+, Cl- (Chloride Ions), K+ (Potassium Ions), Na+ (Sodium Ions) and this process is affected in failing hearts. Ca2+ uptake activates Caln (Calcineurin), which further facilitates NFAT (Nuclear Factor of Activated T-Cells) translocation to the nucleus, a process that is quite essential for axonal growth. cAMP plays a vital role in regulation of cardiovascular function by controlling the process of myocardial contraction. Increase in higher concentration of Ca2+ and PKA activation enhances eNOS (Endothelial NOS) enzyme activity by phosphorylation of Serine residue (Ser635) in order to stimulate eNOS signaling, which is essential to maintain cardiovascular homeostasis (Ref.13). In mammals, Ca2+ and HCO3- play a critical role in the regulation of sperm function, most likely by regulation of cAMP levels. sAC is active in human spermatozoa and is a sensor for both HCO3- and Ca2+. Ca2+ release by CaCn and CNG, activate Calm (Calmodulin) and CamKs (Calcium/Calmodulin-Dependent Protein Kinases). Calm further activate Caln, CamK2 (Calcium/Calmodulin-Dependent Protein Kinase-II) and CamK4. These in turn modulate cAMP production by regulating the activity of ACs and PDEs. The CamKs along with Caln inhibit PDE and ACIX, whereas CamK2 and CamK4 inhibit the function of ACIX and ACI, respectively (Ref.2). ACIX is also inhibited by PKC thus controlling cAMP signaling in the hippocampal neurons. cAMP activated PKA represses ERK activation by the formation of an inactive Rap1/Raf1 that interferes with ERK activation through seizure of MEK1/2 activation by sequestering Raf1 activity. 14-3-3 like the PKA also aids the process of Raf1 inactivation. Therefore, the effect of cAMP on ERK differs depending on the balance of the Raf1, BRaf and PKA isoforms occurring inside a cell (Ref.10). Long PDE4A (cAMP Specific Phosphodiesterase-4A) isoforms are activated by cAMP, PKA and Akt kinases. The PDE sequester cAMP activity by converting it back to AMP. This negative-feedback loop terminates the cAMP signal locally. cAMP further represses the activity PDE1 (Phosphodiesterase-1) to enhance the duration and intensity of cAMP signaling (Ref.14).
cAMP follows a distinct route and activates a single PKA-AKAP complex close to the substrate to mediate a distinct biological effect. Accordingly, each substrate appears to have its own, private anchored pool of PKA and its own local gradient of cAMP. PKA inhibit the interaction of 14-3-3 proteins with BAD (through 14-3-3/BAD signaling) and NFAT to promote cell survival. PKA inhibits Adducin action by limiting its role during assembly of Spectrin-Actin network in erythrocytes, thereby reducing the chances of Erythroleukemia. It activates KDELR (KDEL (Lys-Asp-Glu-Leu) Endoplasmic Reticulum Protein Retention Receptor) to promote retrieval of proteins (protein retention) from golgi complex to endoplasmic reticulum thereby maintaining steady state of the cell. Increased cAMP levels promote survival of neuronal cells by inactivating GSK3Alpha (Glycogen Synthase Kinase-3-Alpha) and GSK3Beta (Glycogen Synthase Kinase-3-Beta) via a PKA dependent mechanism and thus prevents Oncogenesis and neurodegeneration (Ref.13 & 15). PKA interferes at different levels with other signaling pathways. Inactivation of PTP (Protein Tyrosine Phosphatase) results in dissociation from and consequent activation of ERKs. Inactivation of PCTK1 (PCTAIRE Protein Kinase-1) and APC (Anaphase-Promoting Complex) helps to maintain control cell proliferation and anaphase initiation and late mitotic events, respectively, thereby checking the degradation cell cycle regulators. PKA activation by cAMP enhances release of stored energy in cells by phosphorylation of HSL (Hormone-Sensitive Lipase) in white adipose tissue, which leads to the hydrolysis of triglycerides (vital intermediates of Tricaylglycerol Metabolism). Hydrolysis of triglycerides by HSL generates free fatty acids, the major gateway for the release of stored energy, and this process is termed as Lipolysis. Gcg binds to GcgR/GN-Alpha proteins on the surface of liver cells and triggers an increase in cAMP production leading to an increased rate of Glycogenolysis by activating PHK (Phosphorylase Kinase) via the PKA-mediated cascade. PHK further activate PYG (Glycogen Phosphorylase), which converts Glycogen to Glucose-1-Phosphate. Phosphoglucomutase then transfers phosphate to C-6 of Glucose-1-Phosphate generating Glucose-1,6-Phosphate as an intermediate. The phosphate on C-1 is then transferred to the enzyme regenerating it and Glucose-6-Phospahte is the released product that enters Glycolysis. This is the same response hepatocytes have to Epinephrine release through the ADR-Alpha/Beta. PKA further inhibits GYS (Glycogen Synthase) leading to seizure of energy consuming process like Glycogen Synthesis (Ref.13 & 16).
Upon stimulation from hormones, cAMP increases phosphorylation of RhoA that inactivates Rho Kinase. Rho Kinase regulates Myosin-II and cell contraction by catalyzing phosphorylation of the regulatory subunit of Myosin phosphatase, PPtase1 (Protein Phosphatase-1), by inhibiting its catalytic activity, which results in an indirect increase in RLC (Regulatory Light Chain of Myosin) phosphorylation. Inactivation of Rho Kinase also directly increases RLC phosphorylation. Such increased intracellular cAMP and PKA activation on RLC phosphorylation decreases Thrombin-induced isometric tension development in endothelial cells and this decrease the development of Edema, a hallmark of acute and chronic inflammation (Ref.17). Interestingly, PKA controls phosphatase activity by phosphorylation of specific PPtase1 inhibitors, such as DARPP32 (Dopamine-and cAMP-Regulated Phosphoprotein). Neurotransmitters enhance DARPP32 interaction via GPCRs, which leads to suppression of PPtase1 activity, when DARPP32 is phosphorylated at Thr-34 (Threonine-34) position. Phosphorylation is a crucial event in transcriptional activation by CREB (cAMP Response Element-Binding Protein), CREM (cAMP Response Element Modulator) and ATF1 (Activating Transcription Factor-1), because it allows interaction with the transcriptional co-activators CBP (CREB-Binding Protein) and p300. CREM gene encodes many different isoforms, some of which have repressive functions. Particularly the repressor ICER (Inducible cAMP Early Repressor), participates in the downregulation of cAMP-induced transcription by competing with the binding of CREB and CREM activators to their DNA binding sites. PPtase1 checks the phosphorylation events in order to inactivate the formation of repressor isoforms like ICER so that CREB, CREM and ATF1 are able to interact with the co-activators like CBP and p300. Hence, under physiological conditions, ICER induction is a transient phenomenon that allows cAMP signaling to return to the basal state. In contrast, prolonged or inappropriate induction of ICER elicits pathological consequences (Ref.18). Similarly, phosphorylation of NF-KappaB (Nuclear Factor-KappaB) by PKA is necessary for transcriptional activation and interaction with CBP. PKA modulates the activity of transcription factors, such as nuclear receptors and HMG (High Mobility Group)-containing proteins, influencing their dimerization or DNA-binding properties. A peculiar example is the mechanism by which PKA regulates Gli3 (Gli-Kruppel Family Member-3) under the influence from Hedgehog signaling. Function of Gli3 is similar to that of Drosophila gene CI (Cubitus Interruptus) activity. In this case phosphorylation stimulates a specific cleavage of Gli3 which transforms the protein from an activator to a repressor. However, proteins like PKIs (Protein Kinase Inhibitors) and Mep1B (Meprin-A-Beta) down regulate PKA activity to prevent aberrant gene expression (Ref.13 & 16).
Normally, the level of intracellular cAMP is regulated by the balance between the activities of two types of enzymes; ACs and the cyclic nucleotide PDEs chiefly in response to hormones and neurotransmitters. The cAMP signaling is involved in controlling exocytotic events in polarized epithelial cells with implication for Diabetes Insipidus, Hypertension, Gastric ulcers, Thyroid disease, Diabetes Mellitus, and Asthma. Heterologous sensitization of cAMP signaling contributes to fundamental physiological processes such as the timing of circadian rhythms, sexual behavior, and neurotransmitter crosstalk, and also to neurological disorders such as substance abuse and drug-induced Dyskinesias (Ref.19). This provide insight into the mechanisms of addiction and many other central nervous system disorders reflective of altered neuronal cell functioning. Besides it also represent very likely roles for ACs and open up avenues for therapeutics targeting ACs that may prove useful in the development of male contraceptives (Ref.1 & 20).