Activation via G-proteins

There are several types of guanylyl cyclase. We know of membrane-located guanylyl cyclases that are activated via G-proteins, and we also know of purely cytoplasmically localized guanylyl cyclases. Regulators of activity of cytoplasmic guanylyl cyclase include NO. 

Activation of G-protein-coupled receptors. There are many routes by which ligandbinding to G-protein-coupled receptors can transmit signals to the ERK pathway (review Liebmann, 2001), and activation of the ERK pathway is frequently observed upon ligand binding to G-protein-coupled receptors. A main entry point is the Raf kinase, which can be activated by protein kinase C and inhibited by protein kinase A. As outlined in Sections 7.3 and 7.4, both enzyme families can be activated via G-protein-signaling pathways by multiple mechanisms. 

Typically, PI-3-kinases are composed of regulatory and catalytic subunits. Activation of them involves binding of the catalytic subunits to activated Ras and, depending on the particular enzyme, binding of the regulatory subunit to phosphotyrosines on activated receptor tyrosine kinases through its SH2 domains or activation via G protein-coupled receptors. 

The antimuscarinic drug atropine, and its derivative ipratropiumbromide, can also be used for antiarrhyth-mic treatment. Muscarinic receptors (M2 subtype) are mainly present in supraventricular tissue and in the AV node. They inhibit adenylylcyclase via G proteins and thereby reduce intracellular cAMP. On the other hand, activation of the M2 receptor leads to opening of hyperpolarizing Ik.acii and inhibits the pacemaker current If probably via the (3y-subunit of the Gi protein associated with this receptor. The results are hyperpolarization and slower spontaneous depolarization. Muscarinic receptor antagonists like atropine lead to increased heart rate and accelerated atrioventricular conduction. There are no or only slight effects on the ventricular electrophysiology. 

The ETa receptor activates G proteins of the Gq/n and G12/i3 family. The ETB receptor stimulates G proteins of the G and Gq/11 family. In endothelial cells, activation of the ETB receptor stimulates the release of NO and prostacyclin (PGI2) via pertussis toxin-sensitive G proteins. In smooth muscle cells, the activation of ETA receptors leads to an increase of intracellular calcium via pertussis toxin-insensitive G proteins of the Gq/11 family and to an activation of Rho proteins most likely via G proteins of the Gi2/i3 family. Increase of intracellular calcium results in a calmodulin-dependent activation of the myosin light chain kinase (MLCK, Fig. 2). MLCK phosphorylates the 20 kDa myosin light chain (MLC-20), which then stimulates actin-myosin interaction of vascular smooth muscle cells resulting in vasoconstriction. Since activated Rho... 

Secondly, treatment of neutrophils with pertussis toxin, which ADP-ribosylates a neutrophil G protein and causes a loss of cell responsiveness via receptor-mediated pathways (40,41), has minimal effect on the response to HCH (Figure 11, lower panel). Thus it can be concluded that HCH activation of neutrophils is independent of receptor-mediated activation of G proteins. 

Figure 6.1. Regulation of adenylate cyclase activity by G-proteins. Occupancy of receptors such as the /3-adrenergic receptor result in the activation (+) of adenylate cyclase via coupling through stimulatory G-proteins (Gs). Alternatively, occupancy of receptors such as the 2-adrenergic receptor inhibit (-) adenylate cyclase via coupling through inhibitory G-proteins (Gj).

The sequences of events that occur during activation of adenylate cyclase after receptor occupancy are shown in Figure 6.3. This scheme thus shows activation of a Gofc-type protein (i.e. a process that leads to the activation of adenylate cyclase), whereas similar processes will occur with a Ga protein, except that the interaction with adenylate cyclase will result in its inactivation. In the same way, activation of phospholipases by mobile Ga-type subunits will occur via similar mechanisms. In the unstimulated state, Gas (or Gcq) is bound to GDP. Binding of the receptor with its agonist induces a conformational change in the receptor that activates its G-protein. This stim-... 

Cockcroft, S., Stutchfield, J. (1989). The receptors for ATP and fMet-Leu-Phe are independently coupled to phospholipases C and A 2 via G-protein(s). Relationship between phospholipase C and A2 activation and exocytosis in HL60 cells and human neutrophils. Biochem. J. 263, 715-23. 

Recently there has been much interest in the possible role of the family of protein kinases which translate information from the second messenger to the membrane proteins. Many of these kinases are controlled by free calcium ions within the cell. It is now established that some serotonin (5-HT) receptors, for example, are linked via G proteins to the phosphatidyl inositol pathway which, by mobilizing membrane-bound diacylglycerol and free calcium ions, can activate a specific protein kinase C. This enzyme affects the concentration of calmodulin, a calcium sequestering protein that plays a key role in many intracellular processes. 

Odorants are thought to bind to integral membrane receptors on the cilia of the olfactory sensory neurons. The receptors are thought to he specific different olfactory neuron types recognize different odorants that share certain characteristics (Buck, 1993). The odorant receptors transduce signals via interactions with G-proteins (so-called because guanosine trisphosphate is involved in their activation). These G-protein-coupled exhibit seven hydrophobic domains (Fig. 5.6). Variation in the amino acid sequence of the transmembrane domain may account for specificity and selectivity of odor reception. 

There are various G-proteins that differ mainly with regard to their a-unit Association with the receptor activates the G-protein, leading in turn to activation of another protein (enzyme, ion channel). A large number of mediator substances act via G-protein-coupled receptors (see p. 66 for more details). 

The effects of insulin on transcription are shown on the left of the illustration. Adaptor proteins Crb-2 and SOS ( son of sevenless ) bind to the phosphorylated IRS (insulin-receptor substrate) and activate the G protein Ras (named after its gene, the oncogene ras see p.398). Ras activates the protein kinase Raf (another oncogene product). Raf sets in motion a phosphorylation cascade that leads via the kinases MEK and ERK (also known as MARK, mitogen-activated protein kinase ) to the phosphorylation of transcription factors in the nucleus. 

Initially, phospholipase A2 [1] releases the arachidonate moiety from these phospholipids. TheactivityofphospholipaseA2 is strictly regulated. It is activated by hormones and other signals via G proteins. The arachidonate released is a signaling substance itself However, its metabolites are even more important. 

Figure 14-1. Signaling via G protein-coupled receptors. Ligand binding to its cell-surface receptor initiates interaction of the receptor with the heterotrimeric G protein for which it is specific. A conformational change in the G protein brought about by binding of the ligand-receptor complex promotes exchange of GDP for GTP. The activated Gd-GTP dissociates from the Gp complex and both can interact with effectors, which carry on the signal to the mechanism that implements the cellular response.

Platelet activation occurs in large part via G protein-coupled agonist receptors and intracellular signaling events that involve activation of phospholipase C (PLC). PLC catalyzes the breakdown of plasma membrane inositol phospholipids, resulting in generation of 1,2-diacylglycerol (DAG) and 1,4,5-inositol triphosphate (IP3). DAG activates protein kinase C, and IP3 induces mobilization of calcium from intracellular stores (10). 

---