Pertussis toxin sensitivity

Excitation of smooth muscle via alpha-1 receptors (eg, in the utems, vascular smooth muscle) is accompanied by an increase in intraceUular-free calcium, possibly by stimulation of phosphoUpase C which accelerates the breakdown of polyphosphoinositides to form the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 releases intracellular calcium, and DAG, by activation of protein kinase C, may also contribute to signal transduction. In addition, it is also thought that alpha-1 adrenergic receptors may be coupled to another second messenger, a pertussis toxin-sensitive G-protein that mediates the translocation of extracellular calcium. 

Functionally, the Dl-like receptors (Dl, D5) are coupled to the G protein Gas and thus can stimulate adenylyl cyclase. The D2-like receptors (D2, D3, and D4) couple to pertussis toxin sensitive G proteins (Gai/0), and consequently inhibit adenylyl cyclase activity. While the Dl-like receptors almost exclusively signal through Gas-mediated activation of adenylyl cyclase, the D2-like receptors have been reported to modulate the activity of a plethora of signaling molecules and pathways. Many of these actions are mediated through the G(3y subunit. Some of these molecules and pathways include the calcium channels, potassium channels, sodium-hydrogen exchanger, arachidonic acid release, and mitogen-activated protein kinase pathways. 

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... 

The OP group of receptois share common effector mechanisms. All receptois couple via pertussis toxin-sensitive Go and Gi proteins leading to (i) inhibition of adenylate cyclase (ii) reduction of Ca2+ currents via diverse Ca2+ channels (hi) activation of inward rectifying K+ channels. In addition, the majority of these receptors cause the activation of phospholipase A2 (PLA2), phospholipase C 3 (PLC 3), phospholipase D2 and of MAP (mitogen-activated protein) kinase (Table 3). 

Pertussis toxin is produced by the bacterium Bordetella pertussis. It covalently modifies G-proteins of the G/Go family (transfer of a ADP-ribose moiety of NAD onto G-protein a-subunits). ADP-ribosylated G-proteins are arrested in their inactive state and, as a consequence, functionally uncoupled from their respective effectors. Examples for pertussis toxin-sensitive cellular responses include the hormonal inhibition of adenylyl cyclases, stimulation ofK+ channels, inhibition of Ca2+ channels and stimulation ofthe cGMP-phosphodiesterase in retinal rods. 

The 3 isozymes are activated by G protein-coupled receptors through two different mechanisms [2]. The first involves activated a-subunits of the Gq family of heterotrimeric G proteins (Gq, Gn, Gi4, G15/16). These subunits activate the (31, (33 and (34 PLC isozymes through direct interaction with a sequence in the C terminus. The domain on the Gqa-subunit that interacts with the (3 isozymes is located on a surface a-helix that is adjacent to the Switch III region, which undergoes a marked conformational change during activation. The second mechanism of G protein activation of PLC 3 isozymes involves (3y-subunits released from Gi/0 G proteins by their pertussis toxin-sensitive activation by certain receptors. The 3y-subunits activate the 32 and 33 PLC isozymes by interacting with a sequence between the conserved X and Y domains. 

The exact process(es) by which a2-adrenoceptors blunt release of transmitter from the terminals is still controversial but a reduction in the synthesis of the second messenger, cAMP, contributes to this process. a2-Adrenoceptors are negatively coupled to adenylyl cyclase, through a Pertussis toxin-sensitive Gi-like protein, and so their activation will reduce the cAMP production which is vital for several stages of the chemical cascade that culminates in vesicular exocytosis (see Chapter 4). The reduction in cAMP also indirectly reduces Ca + influx into the terminal and increases K+ conductance, thereby reducing neuronal excitability (reviewed by Starke 1987). Whichever of these releasecontrolling processes predominates is uncertain but it is likely that their relative importance depends on the type (or location) of the neuron. 

Guntermann C, Murphy BJ, Zheng R, Qureshi A, Eagles PA, Nye KE. Human immunodeficiency virus-1 infection requires pertussis toxin sensitive G-protein-coupled signalling and mediates cAMP downregulation. Biochem Biophys Res Commun 1999 256(2) 429M35. 

Kaminski NE, Koh WS, Yang KH, Lee M, Kessler FK. Suppression of the humoral immune response by cannabinoids is partially mediated through inhibition of adenylate cyclase by a pertussis toxin-sensitive G-protein-coupled mechanism. Biochem Pharmacol 1994 48 1899-1908. 

BP Hughes, JN Crofts, AM Auld, LC Read, GJ Barritt. (1987). Evidence that a pertussis-toxin-sensitive substrate is involved in the stimulation by epidermal growth factor and vasopressin of plasma-membrane Ca2+ inflow in hepatocytes. Biochem J 248 911-918. 

However, Gi2 is unlikely to be the only G protein that couples 8 receptors to adenylyl cyclase since the cloned 8 receptor expressed in HEK 293 cells effectively couples to adenylyl cyclase via a pertussis toxin sensitive G protein [74], yet these cells lack immunologically detectable Gi2 and G0 [78] suggesting that either Gix or Gi3 in these cells couples the 8 receptor to adenylyl cyclase. Prather et al. [69] and Law and Reisine [73] have reported that 8 receptors can associate with Gix and Gi3. Furthermore, Sanchez-Blazquez et al. [68] have reported that antisense against Gi3 mRNA administered to the nervous system blocked 8 agonist-induced analgesia. 

The selectivity in muscarinic receptor coupling is not, however, absolute. Overexpression of receptors or of particular G proteins supports interactions that may differ from those described above. For example, M2 receptors expressed in Chinese hamster ovary cells not only inhibit adenylyl cyclase but also can stimulate phosphoinositide hydrolysis through a pertussis-toxin-sensitive G protein [52] this is not seen, however, when M2 receptors are expressed in Y1 cells. These findings indicate that caution must be exercised in interpreting data obtained when receptors are expressed, often at high levels, in cells in which they normally do not function. 

Leaney, J. L. and Tinker, A. (2000) The role of members of the pertussis toxin-sensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel. Proc. Natl. Acad. Sci. USA 97, 5651-5656. 

Sawyer, G. W. and Ehlert, F. J. (1999) Muscarinic M3 receptor inactivation reveals a pertussis toxin-sensitive contractile response in the guinea pig colon evidence for M2/M3 receptor interactions../. Pharmacol. Exp. Ther. 289,464-476. 

Cockcroft, S., Stutchfield, J. (1989). ATP stimulates secretion in human neutrophils and HL60 cells via a pertussis toxin-sensitive guanine nucleotide-binding protein coupled to phospholipase C. FEBS Lett. 245,25-9. 

The G-protein that has been termed Gp, and that is linked to phospholipase C activation, may in fact be Gaj 2 or Gc. 3. Ga is designated as the G-protein responsible for activation of phospholipase A2, which results in arachidonic acid release. Some experimental evidence indicates that, at least in HL-60 cells, different agonists can preferentially activate different phospholipases, and some of these are responsible for the activation of secretion. In neutrophils, the two pertussis-toxin-sensitive Ga-proteins (Gaj-2 and G j 3) have been identified by peptide mapping of proteolytic digests of the proteins, by peptide sequencing and by immunoblotting. Complementary-DNA clones for the mRNA of these two molecules have also been isolated from an HL-60 cDNA library. Gai-2 is five to ten times more abundant than Gai.3, the former component comprising 3% of the total plasma membrane proteins. It is possible that these two different Ga-subunits are coupled to different phospholipases (e.g. phospholipases C and D). Pertussis toxin inhibits the secretion of O2 after stimulation of neutrophils by fMet-Leu-Phe, but pertussis-toxin-insensitive G-proteins are also present in neutrophils. These may be members of the Gq family and may be involved in the activation of phospholipase Cp (see 6.3.1). 

The receptors for fMet-Leu-Phe, C5a and PAF have all been cloned (see Chapter 3) and possess the predicted seven membrane-spanning domains present in other G-protein-linked receptors of the rhodopsin superfamily (see Fig. 3.2). The pertussis-toxin sensitivity of the G-proteins associated with these receptors arises from the ADP-ribosylation of a cysteine residue that is four amino acids from the COOH-terminus of the molecule. Some other pertussis-toxin-insensitive G-proteins that exist lack this critical cysteine residue. 

Ca2+ channels Ginseng extract rapidly and reversibly inhibits high-threshold, voltage-dependent Ca2+ channels in micromolar doses (Nah and McCleskey 1994 Nah et al. 1995). This effect is mediated by a receptor linked to a pertussis toxin-sensitive G protein, but it is not through o2 adrenergic, GABAB, muscarinic, or opioid receptors (Nah and McCleskey 1994). Several ginsenosides inhibit Ca2+ currents by 16 to... 

Nah SY, McCleskey EW. (1994). Ginseng root extract inhibits calcium channels in rat sensory neurons through a similar path, but different receptor, as mu-type opioids. J Ethnopharmacol. 42(1) 45-51. Nah SY, Park HJ, McCleskey EW. (1995). A trace component of ginseng that inhibits Ca2+ channels through a pertussis toxin-sensitive G protein. Proc Natl Acad Sci USA. 92(19) 8739-43. 

Nah SY, Park FIJ, McCleskey EW. (1995). A trace component of ginseng that inhibits Ca2+ channels through a pertussis toxin-sensitive G protein. Proc Natl Acad Sci USA. 92(19) 8739-43. 

Sillard, R., Harii, K. and Takuwa, Y., 1999, The novel sphingosine 1-phosphate receptor AGR16 is coupled via pertussis toxin-sensitive and -insensitive G-proteins to multiple signaling pathways. Biochem. J. 337 67-75. 

Idzko M. Dichmann S, Ferrari D, Di Virgilio F. la Sala A, Girolomoni G. Panther E. Norgauer J Nucleotides induce chemotaxis and actin polymerization in immature but not mature human dendritic cells via activation of pertussis toxin-sensitive P2y receptors. Blood 2002 100 925-932. 

MacNulty EE, McClue SJ, Carr IC, Jess T, Wakelam MJ, Milligan G (1992) a2-C10 adrenergic receptors expressed in rat 1 fibroblasts can regulate both adenylylcy-clase and phospholipase D-mediated hydrolysis of phosphatidylcholine by interacting with pertussis toxin-sensitive guanine nucleotide-binding proteins. J Biol Chem 267 2149-2156... 

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