Cytoplasmic Second Messengers

Binding of an agonist to a receptor provides the first message in receptor signal transduction to effector to affect cell physiology. The first messenger promotes the cellular production or mobilization of a second messenger, which initiates cellular signaling through a specific biochemical pathway. Physiological signals are integrated within the cell as a result of interactions between and among second-messenger pathways. Compared with the number of receptors and cytosolic signaling proteins, there are relatively few recognized cytoplasmic second messengers. However, their synthesis or release and degradation or excretion reflects the activities of many pathways. Well-studied second messengers include cyclic AMP, cyclic GMP, cyclic ADP—ribose, Ca2+, inositol phosphates, diacylglycerol, and nitric oxide (NO). Second messengers influence each other both directly, by altering the other's metabolism, and indirectly, by sharing intracellular targets. This pattern of regulatory pathways allows the cell to respond to agonists, singly or in combination, with an integrated array of cytoplasmic second messengers and responses.

Cyclic AMP

Cyclic AMP, the prototypical second messenger, is synthesized by adenylyl cyclase under the control of many GPCRs; stimulation is mediated by Gs; inhibition, by G. There are nine membrane-bound isoforms of adenylyl cyclase (AC). The membrane-bound ACs are 120 kDa glycoproteins with six membrane-spanning helices; and two large cytoplasmic domains. Membrane-bound ACs exhibit basal enzymatic activity that is modulated by binding of GTP-liganded a subunits of stimulatory and inhibitory G proteins (G and G). ACs are catalogued based on their structural homology and their distinct regulation by G protein a and f3y subunits, Ca2+, protein kinases, and the actions of the diterpene forskolin. Because each AC isoform has its own tissue distribution and regulatory properties, different cell types respond differently to similar stimuli.

The role of drugs interacting at GPCRs as agonists is to accelerate the exchange of GDP for GTP on the a subunits of these G proteins. Once activated by as-GTP, AC remains activated until as hydrolyzes the bound GTP to GDP, which returns the system to its ground state. A single AC activation produces many molecules of cyclic AMP, which, in turn, can activate PKA. Cyclic AMP is eliminated by a combination of hydrolysis, catalyzed by cyclic nucleotide phosphodiesterases, and extrusion by several plasma membrane transport proteins.

Phosphodiesterase

Phosphodiesterases (PDEs) are regulated by controlled transcription as well as by second messengers (cyclic nucleotides and Ca2+) and interactions with other signaling proteins such as b-arrestin and protein kinases. PDEs are responsible for the hydrolysis of the cyclic 3',5'-phosphodiester bond found in cyclic AMP and cyclic GMP. PDEs comprise a superfamily with 11 subfamilies distinguished on the basis of amino acid sequence, substrate specificity, pharmacological properties, and allosteric regulation. The substrate specificities of the PDEs include enzymes that are specific for cyclic AMP, cyclic GMP, and both. PDEs play a highly regulated role that is important in controlling the intracellular levels of cyclic AMP and cyclic GMP. The importance of the PDEs as regulators of signaling is evident from their development as drug targets in diseases such as asthma and chronic obstructive pulmonary disease, cardiovascular diseases such as heart failure and atherosclerotic peripheral arterial disease, neurological disorders, and erectile dysfunction.

Cyclic GMP

Cyclic GMP is generated by two distinct forms of guanylyl cyclase (GC). NO stimulates soluble guanylyl cyclase (sGC), and the natriuretic peptides, guanylins, and heat-stable Escherichia coli enterotoxin stimulate members of the membrane-spanning GCs (e.g., particulate GC).

Actions of Cyclic Nucleotides

In most cases, cyclic AMP functions by activating the isoforms of cyclic AMP-dependent protein kinase (PKA), and cyclic GMP activates a PKG. Recently, a number of additional actions of cyclic nucleotides have been described, all with pharmacological relevance.

Cyclic Nucleotide-Dependent Protein Kinases

PKA holoenzyme consists of two catalytic (C) subunits reversibly bound to a regulatory (R) subunit dimer. The holoenzyme is inactive. Binding of four cyclic AMP molecules, two to each R subunit, dissociates the holoenzyme, liberating two catalytically active C subunits that phosphorylate serine and threonine residues on specific substrate proteins.

PKA diversity lies in both its R and C subunits. Molecular cloning has revealed a and b iso-forms of both the classically described PKA regulatory subunits (RI and RII), as well as three C subunit isoforms Ca, Cb, and Cg. The R subunits exhibit different binding affinities for cyclic AMP, giving rise to PKA holoenzymes with different thresholds for activation. In addition to differential expression of R and C isoforms in various cells and tissues, PKA function is modulated by subcellular localization mediated by A-kinase-anchoring proteins (AKAPs).

PKA can phosphorylate both final physiological targets (metabolic enzymes or transport proteins) and numerous protein kinases and other regulatory proteins in multiple signaling pathways. This latter group includes transcription factors that allow cyclic AMP to regulate gene expression in addition to more acute cellular events.

Cyclic GMP activates a protein kinase, PKG, that phosphorylates some of the same substrates as PKA and some that are PKG-specific. Unlike PKA, PKG does not disassociate upon binding cyclic GMP. PKG is known to exist in two homologous forms. PKGI, with an acetylated N terminus, is associated with the cytoplasm and known to exist in two isoforms (Ia and Ib) that arise from alternate splicing. PKGII, with a myristylated N terminus, is membrane-associated and may be compartmented by PKG-anchoring proteins in a manner similar to that known for PKA. Pharmacologically important effects of elevated cyclic GMP include modulation of platelet activation and regulation of smooth muscle contraction.

Cyclic Nucleotide-Gated Channels

In addition to activating protein kinases, cyclic AMP and cyclic GMP also bind to and directly regulate the activity of plasma membrane cation channels referred to as cyclic nucleotide-gated (CNG) channels. CNG ion channels have been found in kidney, testis, heart, and the CNS. These channels open in response to direct binding of intracellular cyclic nucleotides and contribute to cellular control of the membrane potential and intracellular Ca2+ levels. The CNG ion channels are multisub-unit pore-forming channels that share structural similarity with the voltage-gated K+ channels.

Calcium

The entry of Ca2+ into the cytoplasm is mediated by diverse channels: Plasma membrane channels regulated by G proteins, membrane potential, K+ or Ca2+ itself, and channels in specialized regions of endoplasmic reticulum that respond to IP3 or, in excitable cells, to membrane depolarization and the state of the Ca2+ release channel and its Ca2+ stores in the sarcoplasmic reticulum. Ca2+ is removed both by extrusion (Na+—Ca2+ exchanger and Ca2+ ATPase) and by reuptake into the endoplasmic reticulum (SERCA pumps). Ca2+propagates its signals through a much wider range of proteins than does cyclic AMP, including metabolic enzymes, protein kinases, and Ca2+-binding regulatory proteins (e.g., calmodulin) that regulate still other ultimate and intermediary effectors that regulate cellular processes as diverse as exocytosis of neuro-transmitters and muscle contraction. Drugs such as chlorpromazine (an antipsychotic agent) are calmodulin inhibitors.

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