Cho

Figure 4.10. Topographicalrepresentation of primary sequence of human j32-adrenergic receptor, a typical G-protein-coupled receptor. The receptor protein is illustrated as possessing seven hydrophobic regions each capable of spanning the plasma membrane, thus creating intracellular and extracellular loops as well as an extracellular amino terminus and a cytoplasmic carboxyl terminal region.

Figure 4.10. Topographicalrepresentation of primary sequence of human j32-adrenergic receptor, a typical G-protein-coupled receptor. The receptor protein is illustrated as possessing seven hydrophobic regions each capable of spanning the plasma membrane, thus creating intracellular and extracellular loops as well as an extracellular amino terminus and a cytoplasmic carboxyl terminal region.

because in addition to the muscarinics, the primary structures of the adrenergic (a,, a„ /3U and fi^), serotonergic, tachykinin, and rho-dopsin receptors have been determined (174176). All of these display the now-familiar homology pattern of seven membrane-spanning domains packed into antiparallel helical bundles (Fig. 4.10). The exceedingly high homology among the large family cf G protein-coupled receptors also has allowed the development of three-dimensional models of the proteins to aid in drug refinement (177). For example, mutagenesis studies on the j32-adrenergic receptor have localized the intracellular domains involved in (l)the couplingof the receptor to G proteins (178); (2) homologous desensitization by j3-adrenergic receptor kinase (/3-ARK) (179), itself cloned and a possible target for down-regulation inhibitors (180); (3) heterologous desensitization by cAMP-dependent protein kinase (181); and (4) an extracellular domain with conserved c teine residues implicated in agonist ligand binding (182). A chimeric muscarinic cholin-

Desensitized

Desensitized

R*L

Figure 4.11. Receptor G-protein-mediated signal transduction, (a) Receptor (R) associates with a specific ligand (L), stabilizingan activated form of the receptor OR*), which can catalyze the exchange of GTP for GDP bound to the a-subunit of a G-protein. The jSy-heterodimer may remain associated with the membrane through a 20-carbon isoprenyl modification of the y-subunit. The receptor is desensitized by specific phosphorylation (-P). (b) The G protein cycle. Pertussis toxin (PTX) blocks the catalysis of GTP exchange by receptor. Activated a-subunits (aGTP) and /3y-heterodimers can interact with different effectors CE). Cholera toxin (CTX) blocks the GTPase activity of some a-subunits, fixing them in an activated form.

Effectors Phosphodiesterase

Phospholipase C Adenylyl cyclase Phospholipase A2

Ion channel

Figure 4.11. Receptor G-protein-mediated signal transduction, (a) Receptor (R) associates with a specific ligand (L), stabilizingan activated form of the receptor OR*), which can catalyze the exchange of GTP for GDP bound to the a-subunit of a G-protein. The jSy-heterodimer may remain associated with the membrane through a 20-carbon isoprenyl modification of the y-subunit. The receptor is desensitized by specific phosphorylation (-P). (b) The G protein cycle. Pertussis toxin (PTX) blocks the catalysis of GTP exchange by receptor. Activated a-subunits (aGTP) and /3y-heterodimers can interact with different effectors CE). Cholera toxin (CTX) blocks the GTPase activity of some a-subunits, fixing them in an activated form.

ergic:p-adrenergic receptor engineered to activate adenylyl cyclase (a second messenger system not coupled to MAChR agonism) also has helped identify which intracellular loops nay be involved in direct G-protein interac-i tions (183). The diverse signal transduction functional roles of the many G-proteins to which these receptors are coupled (Fig. 4.11) also makes them viable drug targets (184).

The complicated biochemical pharmacology of natriuretic peptides, the regulatory system that ads to balance the renin-angiotensin-aldosterone system (185), has been significantly clarified by the cloning of three receptor subtypes, which revealed the functional characteristics cf a new paradigm for second messenger signal transduction through guanylate cyclase. The a-atrial natriuretic peptide (a-ANP) receptor (NPA-R) and the brain natriuretic peptide (BNP) receptor (NPB-R) contain both protein kinase and guanylate cyclase (GC) do mains, as determined by both sequence homologies and catalytic activities, whereas the clearance receptor (ANP-C) completely lacks the necessary intracellular domains for signal transduction through the guanylate cyclase pathway (186). This system defines the first example of a cell surface receptor that enzymat-ically synthesizes a diffusible second messenger system in response to hormonal stimulation (187) (Fig. 4.12). Data from experiments performed with C-ANP4_23 indicates that the clearance receptor (NPC-R) may be coupled to the adenylate cyclase/cAMP signal transduction system through an inhibitory guanine nucleotide regulatory protein (188). Because the NPs have differential, but not absolute, affinities for their corresponding receptors (189) and because both agonism (190) and antagonism (191) of the GC activity have been demonstrated in vitro using ANP analogs, it may be possible to discriminate among the receptor

Figure 4.12. Model for ANP-A and ANP-B receptor function. The unoccupied ANP-A receptor is shown on the left with a basal rate of cGMP synthesis (indicated by a thin arrow). The effect of ligand binding to the amino-terminal extracellular domain is shown on the right. Proposed allosteric modulation of guanylate cyclase by a-ANP is schemat ically illustrated by a change in shape of the intracellular domain and a thicker arrow to denote an increase in guany-late cyclase-specific activity with greater production of the second messenger cGMP.

cGMP + PPi

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