SBC 302: LECTURE 8-cAMP AND CELL SIGNALLING

COUPLING OF CELL SURFACE RECEPTORS TO INTRACELLULAR SIGNALING

In order for the many hormones that bind exclusively to the outer surface of cells to carry

out their actions, there must be some means of translating the extracellular signal into an

intracellular response. The first example of a trans duction system that was understood in

some detail derived from investigating one of the key features of the fight of flight

response, the mobilization of stoned carbohydrate in the liver. The physiologic response to

stress requires a supply of readily consumable Energy, best provided in the form of blood

glucose, which is stored as the polysaccharide glycogen at the highest levels in the liver. β-

Adrenergic stimulation of hepatocytes by epinephrine leads rapidly to the hydrolysis of

glycogen and the release of free sugar glucagon also stimulates the breakdown of hepatic

glycogen. The mechanism used to transmit this response is the prototypical example of a

second messenger system, in which the agonist that interacts with the outside of the cell in

this case glucagon or epinephrine, is considered a first messenger, and a soluble,

intracellular signaling molecule generated by hormone-receptor association is called a

Second first messenger. According to this model, there is no need for the hormone to enter

the cell all that is required is a receptor for the hormone and an apparatus to trans duce

receptor occupancy into the generation of a secondary intracellular signal. For hepatic

glycogen breakdown in response to glucagon or β-Adrenergic agents, the second

messenger is CAMP, which is produced by a plasma membrane enzyme, adenylyl cyclase,

from ATP. Adenylyl cyclase is a direct target of Gs which becomes GTP-loaded and active in

response to receptor occupancy CAMP is degraded to AMP and phosphate by a specific PDE

and the balance between these two activities determines the levels of the cyclic nucleotide.

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The scope and diversity of hormones and other extra cellular signals that activate adenylyl

cyclase and increase the level of intracellular CAMP are remarkably extensive. Included in

the long list of hormones that signal through this mechanism are B-adrenergic agents,

glycoprotein hormones such as TSH, glucagon, adrenocorticotropic hormone (ACTH),

hypothalamic hormones, and antidiuretic hormone. Moreover, the range of physiologic and

bio. chemical events modulated by CAMP is equally vast. Thus, although the second

messenger CAMP defines a commonly used mechanism for transducing signals from

extracellular hormones, it also presents another problem in signaling: how do cells

maintain selectivity in how they respond to a given hormone? Much of this is accomplished

by the subcellular compartmentalization of signaling complexes A-kinase anchoring

proteins (AKAPS), which are scaffolds localized to distinct intracellular sites, bind a

number of proteins that modulate the actions of CAMP including degrading enzymes and

target kinases. The regulated assembly of higher order structures confers a spatiotemporal

resolution to CAMP signaling that can allow multiple biologic responses to exist within the

same cell.

cAMP Signaling in Eukaryotes

In eukaryotes, cAMP acts as a secondary messenger, produced in response to extracellular

stimuli and is used to trigger a variety of intracellular responses. In mammalian cells the

stimulus is usually a hormone or neurotransmitter but in yeast there is evidence for

metabolites acting as the stimuli. The extracellular response is usually detected by a

membrane receptor which then activates or inhibits adenylate cyclase, increasing and

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decreasing cAMP production, respectively. For example, in mammalian cells, adrenaline

binds to β-adrenergic receptors stimulating cAMP production, whilst acetylcholine binds to

muscarinic receptors inhibiting cAMP production. In the yeast Saccharomyces cerevisiae

the cAMP signal transduction pathway is used to control mating in response to the binding

of a or α pheromones to the complementary Ste2 and Ste3 receptors, and the Gpr1 receptor

responds to nitrogen limitation to induce filamentous growth. Generally, the receptors are

seven-helix membrane proteins that interact with small G-proteins and are termed G-

protein-coupled receptors (GPCR). G-proteins are frequently heterotrimeric proteins

composed of a GTP-binding α-subunit and two intimately associated β and γ subunits,

which have approximate molecular masses of 45 kDa, 35 kDa, and 7 kDa, respectively. The

binding of GTP activates the G-protein, which subsequently adopts an inactive form as the

GTP is hydrolyzed to GDP. The interaction of the receptor with the inactive G-protein–GDP

complex triggers exchange of the GDP for GTP, activating the α-subunit. The α-subunit

dissociates from the β/γ regulatory subunits and interacts with adenylate cyclase,

increasing or decreasing its activity to effect changes in the concentration of cAMP.

Interestingly, only an α-subunit mediates the signal of the filamentous growth pathway in

S. cerevisiae (e.g., nitrogen starvation triggers an increase in cAMP, which then induces the

cells to produce pseudohyphae and to grow invasively into the agar medium support). In

addition, the G-protein Ras, which commonly activates MAP kinase signal transduction

pathways, can also activate adenylate cyclase in yeast.

Adenylate cyclase is an integral membrane protein, with a molecular mass of

approximately 120 kDa. It has a topology that consists of two membrane domains, each

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composed of six α-helices, and two cytoplasmic catalytic domains, one connecting the two

membrane domains and the other at the C-terminal end of the protein. Adenylate cyclase

deactivates the Gα protein by stimulating its GTPase activity. Increases in cAMP levels are

downregulated by phosphodiesterases (PDEs), which convert cAMP to AMP.

cAMP elicits its effects by binding to protein kinase A (PKA), a tetrameric protein composed

of two regulatory subunits, which bind cAMP, and two catalytic subunits, which act as

kinases phosphorylating serines/threonines in target proteins containing the consensus

sequence Arg-Arg-X-Ser/Thr-X. Mammalian cells possess three isoforms of the catalytic

subunit (i.e., Cα, Cβ, and Cγ) and two isoforms of the regulatory subunits (i.e., RI and RII).

Differences in the regulatory subunits determine the cellular location of the PKA; while

some are cytoplasmic, others are associated with cellular structures and organelles owing

to an interaction between the regulatory (e.g., RII) subunit and A-kinase anchoring proteins

(AKAPs). Owing to a dimerization domain at the N-terminus, the regulatory subunits exist

as a dimer. Each subunit also includes a hinge region, toward the N-terminus, and two

structurally and kinetically distinct cAMP binding sites in the C-terminal end, which

probably arose by a gene-duplication event. The hinge region, which is highly susceptible

to proteolytic cleavage, includes a consensus phosphorylation sequence and acts as a

pseudosubstrate site to which the catalytic domain binds with high affinity. This then acts

as an autoinhibitory domain, with the type II site differing from the type I in that it has a

phosphorylatable serine. Although S. cerevisiae possesses three different catalytic subunits,

it only has a single regulatory subunit and no AKAPs have been identified on sequencing

the genome. The binding of cAMP to the regulatory subunits causes dissociation of these

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subunits from their complex with the catalytic subunits, which can then phosphorylate

downstream target proteins to alter their activity. For example, the hormone adrenaline

controls glycogen metabolism via phosphorylation of phosphorylase kinase and glycogen

synthase by PKA. Phosphorylase kinase activates phosphorylase, an enzyme that breaks

down glycogen, releasing glucose for metabolism, while phosphorylation of glycogen

synthase inhibits the synthesis of glycogen. There are hundreds of known physiological

substrates of PKA, including metabolic enzymes, hormone receptors, and ion channels. PKA

can also regulate gene transcription by phosphorylating target transcription factors, such

as CREB in mammals and Flo8 in S. cerevisiae. CREB binds to cAMP-response elements

found in the promoter regions of a number of genes, such as those encoding enzymes

involved in gluconeogenesis. Flo8 controls the transcription of the cell-surface flocculin,

Flo11 – a class of serine/threonine-rich glycosylphosphatidyl-inositol-anchored cell wall

proteins that have a role in the calcium-dependent process of cell–cell adhesion known as

flocculation. Moreover, Flo11 plays a critical role in the production of pseudohyphae by,

and invasive growth of, S. cerevisiae in response to nitrogen starvation, as the cells

probably search for a new nutrient source. The reassociation of the PKA tetramer is driven

by phosphatases that phosphorylate the RII subunit and by the binding of MgATP to the RI

subunit.

cAMP can also interact with channels that conduct monovalent and divalent cations. The

channel is composed of four or five subunits, with each subunit adopting a six-helix

topology in which there is a pore-forming segment coupling helices 5 and 6. There is a

single cAMP binding-site in the C-terminal end of each subunit, which triggers channel

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opening on binding cAMP. The degree of occupancy of the multiple cAMP binding sites

present within a channel may regulate its conductance.

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Figure: Cyclic AMP is synthesized from ATP by the action of the enzyme adenylyl

cyclase. Binding of the hormone to its receptor activates a G protein which, in turn,

activates adenylyl cyclase. The resulting rise in cAMP turns on the appropriate

response in the cell by either (or both): changing the molecular activities in the

cytosol, often using Protein Kinase A (PKA) — a cAMP-dependent protein kinase that

phosphorylates target proteins turning on a new pattern of gene transcription.