Signal Transduction via Membrane Receptors

Signal transduction across a cell membrane and activation of signaling pathways within the cell are diverse and complex processes. Indeed, even a superficial description of the many pathways is beyond the scope of this chapter. Hence, discussion primarily will be limited to receptors whose transduction mechanisms involve guanine-nucleotide binding proteins (G proteins), "direct" activation of protein kinases, or "direct"activation of proteases. It will become apparent that there is considerable overlap among all signaling pathways.

4.4.1 G-protein-coupled receptors (GPCR)

The prototypical GPCR comprises seven a-helices, each of which crosses the cell membrane (i.e., a 7-transmembrane receptor). The a-helices are connected by peptide sequences (loops) that contain binding sites for ligands and signaling molecules (Fig. 4.4). The extracellular portion of the receptor contains a ligand-binding site while the intracellular loops associate with heterotrimeric G proteins composed of a-, b-, and g-subunits. G-protein subunits activated by ligand binding commonly couple the receptor to

Figure 4.4 The figure shows a prototypical G-protein-coupled receptor (GPCR). GPCRs are characterized by seven transmembrane helices (H1-H7) connected by intervening loops. Extracellular loops contain a ligand-binding site (L) and intracellular loops bind a G protein composed of a, //, and g subunits. For details, see Sec. 4.4.1.

Figure 4.4 The figure shows a prototypical G-protein-coupled receptor (GPCR). GPCRs are characterized by seven transmembrane helices (H1-H7) connected by intervening loops. Extracellular loops contain a ligand-binding site (L) and intracellular loops bind a G protein composed of a, //, and g subunits. For details, see Sec. 4.4.1.

enzymes, ion channels, or other effector molecules in the cell. Signaling through GPCRs can be quite complex, because there are many different G proteins that interact to regulate multiple pathways within a cell.

Activation of heterotrimeric G proteins primarily involves an exchange by Ga of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) and dissociation of Ga from the /g subunits. This GDP-GTP "off-on" switch mechanism is encountered in many regulatory pathways that incorporate small GTP-binding proteins (proteins related to Ga) that belong to five subfamilies (i.e., Ras, Rho, Ran, Rab, and Arf). Ga remains in an activated state until GTPase activity intrinsic to Ga hydrolyzes the GTP to GDP. For some time, it was believed that the primary function of the /g subunits was inhibition of Ga. However, it has been shown that the /g subunits also exert important intracellular actions. One example discussed in Sec. 4.2 on Receptor Binding was that released /g subunits stimulate a GPCR kinase (GRK) that phosphorylates the receptor. The phosphorylated receptor then binds regulatory proteins called /-arrestins which induce down-regulation.

The adenylyl cyclase (AC)-cAMP pathway. One of the first G-protein-coupled pathways to be identified was the adenylyl cyclase (AC)-cAMP pathway (Fig. 4.5). This pathway is activated by many G-protein-coupled receptors (GPCRs) including /-adrenergic receptors that bind epineph-rine (adrenalin) and norepinephrine (noradrenalin). Binding of epi-nephrine to the extracellular portion of its receptor transmits a signal to the associated G-protein (Gs or "stimulatory" G protein) that causes

Figure 4.5 cAMP-adenylyl cyclase (AC) system. Figure shows an activated Ga (i.e., associated with GTP) bound to AC, a transmembrane enzyme. AC activated by Ga converts ATP to the "second messenger" cAMP. Released bg subunits are free to activate additional pathways.

Gsa to exchange GDP for GTP. The activated Gs (Gsa-GTP) dissociates from the bg subunits and binds to adenylyl cyclase (AC), a transmembrane enzyme. AC bound to Gsa-GTP actively converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). Because cAMP mediates some of the actions of the receptor, it often is referred to as a second messenger. Although ingrained in the literature, the term second messenger is of limited value because receptors often generate multiple messengers that cannot be placed in any specific order.

The pathway leading from activation of a GPCR to generation of cAMP may seem more complex than is necessary just to create an intracellular signaling molecule. However, multistep pathways allow for amplification and integration. For example, activation of AC by Gsa-GTP can amplify the original signal by producing numerous molecules of cAMP. Integration is achieved when other pathways modulate (1) Gsa activation, (2) adenylyl cyclase (AC) activation, or (3) accumulation of cAMP.

Activation of Gsa is limited by intrinsic GTPase activity that hydrolyzes bound GTP to GDP. However, proteins known as regulators of G-protein signaling (RGS) can promote GTP hydrolysis and inacti-vation of heterotrimeric G proteins. RGS proteins are similar to the GTPase-activatingproteins (GAPs) (Fig. 4.6) that play important roles in regulating the activities of the closely related small G-proteins. Heterotrimeric G proteins and small G proteins also are regulated by guanine nucleotide exchange factors (GEFs) (Fig. 4.6) that promote exchange of GDP for GTP, and thus promote G-protein activation. The receptor generally serves as the GEF for heterotrimeric G proteins but in some cases, activators of G-protein signaling (AGS) proteins may stimulate GDP-GTP exchange independent of a receptor.

Figure 4.6 Regulation of G-protein activity. G-protein activity can be inhibited by GTPase activating proteins (GAPs) that increase hydrolysis of GTP to GDP and stimulated by guanine nucleotide exchange factors (GEFs) that promote exchange of GDP for GTP (GEFs).

Adenylyl cyclase (AC) and cAMP are also points of integration. For example, a competing pathway may activate an "inhibitory" G protein (Gj) that decreases the activity of AC or activates phosphodiesterases that degrade cAMP to 5'-adenosine monophosphate (5'-AMP). Hence, every step within a single signaling pathway represents a highly controlled intersection in a complex cellular road map.

Cellular signaling by cAMP occurs in large part through activation of protein kinase A (PKA), a serine-threonine kinase. More recently, it has been shown that cAMP also activates PKA-independent pathways through the exchange protein directly activated by cAMP (Epac). Epac is a GEF for the small GTPases Rapl and Rap2 (Ras-proximate-1, 2), small GTP-binding switch proteins that belong to the Ras family. This latter pathway is mentioned to illustrate the complexity of cellular signaling, but not discussed in detail here.

Cyclic AMP activates PKA by binding to the regulatory subunit (R) of the enzyme and releasing the catalytic subunit (C) (Fig. 4.7). The catalytic subunit is then capable of transferring a phosphate group from ATP to serine and threonine residues in specific proteins. Binding of a negatively charged phosphate can alter the conformation and activity of the protein.

Many proteins serve as targets for activated PKA (Fig. 4.8). One example is the cAMP response-element binding protein (CREB). Upon phosphorylation, CREB translocates to the nucleus and binds to DNA at a specific sequence of bases known as the cAMP response element (CRE). CREB then regulates gene transcription by interacting with various coactivators and/or corepressors in a manner similar to the scenario described earlier for the estrogen receptor. Many other proteins also

protein

protein-P

Figure 4.7 Activation of PKA by cAMP. Two molecules of cAMP bind to each of the regulatory subunits (R) of PKA and release the active catalytic subunits (C). The catalytic subunits convert ATP to cAMP.

are phosphorylated by PKA, including enzymes, G-proteins, ion channels, cytoskeletal proteins, and receptors.

Phosphorylation often alters protein function. This was also explained as important covalent modification of enzyme activity (see Sec. 3.4). One of the first PKA-regulated pathways identified was the pathway leading to glycogen breakdown. In this pathway, PKA phosphorylates the enzyme phosphorylase kinase, which in turn phosphorylates the enzyme phosphorylase. Activated (phosphorylated) phosphorylase promotes breakdown of glycogen to glucose. This enzyme cascade allows for signal amplification and provides points for signal integration. Hence, triggering the cAMP-PKA pathway can alter many cytoplasmic and nuclear events within a cell.

CREB ATP protein

CREB-P (transcription factor)

protein-P (enzyme, G-protein, channel cytoskeleton, receptor, etc.)

Activity?

Figure 4.8 The active catalytic subunit (C) of PKA phos-phorylates CREB and many other proteins in the cell. Phosphorylation can alter the activity of the proteins, and thus affect cell function.

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