CAMP formation

A critical component of the G-protein effector cascade is the hydrolysis of GTP by the activated a-subunit (GTPase). This provides not only a component of the amplification process of the G-protein cascade (63) but also serves to provide further measures of dmg efficacy. Additionally, the scheme of Figure 10 indicates that the coupling process also depends on the stoichiometry of receptors and G-proteins. A reduction in receptor number should diminish the efficacy of coupling and thus reduce dmg efficacy. This is seen in Figure 11, which indicates that the abiUty of the muscarinic dmg carbachol [51 -83-2] to inhibit cAMP formation and to stimulate inositol triphosphate, IP, formation yields different dose—response curves, and that after receptor removal by irreversible alkylation, carbachol becomes a partial agonist (68). 

Stimulation and inhibition of the enzyme by the GPCR-G-protein cycle occur by analogous mechanisms. Agonists induce hormone receptors to increase a Ga-GDP-GTP exchange and subsequent Ga 3y dissociation (GDP-a py + GTP GTP-ax + [3y + GDP) (Fig. 4). Consequently, agents that affect either the dissociation of either G or Gs, or the association of their respective as, a , or (3y subunits with adenylyl cyclase could affect rates of cAMP formation in enzyme preparations or in intact cells and tissues. There are several important examples. Gas is stably activated by poorly hydrolyzable analogs of GTP, e.g. GTPyS... 

Adenylyl Cyclases. Table 5 Prodrug inhibition of [3H] cAMP formation in intact cells. Cells were prelabeled for 2 h with [3H]adenine before 50 pM forskolin and pronucleotides were added. After a 15 min incubation the newly formed [3H]cAMP was extracted and quantified as in (7)... 

P-site ligands inhibit adenylyl cyclases by a noncompetitive, dead-end- (post-transition-state) mechanism (cf. Fig. 6). Typically this is observed when reactions are conducted with Mn2+ or Mg2+ on forskolin- or hormone-activated adenylyl cyclases. However, under- some circumstances, uncompetitive inhibition has been noted. This is typically observed with enzyme that has been stably activated with GTPyS, with Mg2+ as cation. That this is the mechanism of P-site inhibition was most clearly demonstrated with expressed chimeric adenylyl cyclase studied by the reverse reaction. Under these conditions, inhibition by 2 -d-3 -AMP was competitive with cAMP. That is, the P-site is not a site per se, but rather an enzyme configuration and these ligands bind to the post-transition-state configuration from which product has left, but before the enzyme cycles to accept new substrate. Consequently, as post-transition-state inhibitors, P-site ligands are remarkably potent and specific inhibitors of adenylyl cyclases and have been used in many studies of tissue and cell function to suppress cAMP formation. 

Preferentially inhibits cAMP formation Preferentially inhibits cAMP formation Preferentially inhibits cAMP formation Preferentially inhibits cAMP formation (in recombinant system). Preferentially inhibits cAMP formation (in recombinant system). 

Effector Gq/11 Preferentially increases Pi hydrolysis and elevates [Ca2+]j Gq/n Preferentially increases Pi hydrolysis and elevates [Ca2+]j (in recombinant systems) Gq/nPreferentially increases Pi hydrolysis and elevates [Ca2+], Intrinsic ligandgated ion channel GsPreferentially increases cAMP formation. 

The 5-HTx receptor class comprises five receptors (5-HT1a, 5-HT1b, 5-HTiD, 5-ht1E> and 5-HT1F) which, in humans, share 40-63% overall sequence identity and couple somewhat preferentially to Gi/o to inhibit cAMP formation (see Tables 1-3). The 5-htiE receptors are given a lower case appellation to denote that... 

G protein effector Gs Gi increases (decreases) cAMP formation not identified Gs preferentially increases cAMP formation Gs preferentially increases cAMP formation... 

HT4, 5-HTreceptor classes because of their limited (<35%) overall sequence identities. This subdivision is arbitrary and may be subject to future modification. 

Inhibition of adenylyl cyclase by mAChR activation results in decreased cAMP formation. A decrease in cAMP is most apparent when adenylyl cyclase is stimulated, for example, by activation of adrenergic receptors... 

The 5-HT4, 5-hts and 5-HT7 receptors are coupled to the stimulation of adenylyl cyclase. 5-HT4, 5-htg and 5-HT7 receptors preferentially couple to the stimulation of adenylyl cyclase, increasing cAMP formation, via the Gs family of G proteins (see Chs 19 and 21). These receptors, however, share only >35% overall sequence homology. For this reason, they are classified as distinct receptor groups or classes and not subtypes of a family. The grouping of these receptors together is considered to be somewhat arbitrary and may be modified in the future. A lower-case appellation is used for the 5-ht6 receptor because a physiological role for these receptors in intact tissue has not been found [28]. 

Regulation of cAMP formation is determined by the activity of adenylyl cyclase 362... 

Adenylyl cyclases are regulated by Gas and Ga. Each of the different forms of adenylyl cyclase can be stimulated by activated Gas (i.e. Gas bound to GTP). In this way Gas couples endocrine and neurotransmitter receptors that stimulate cAMP formation to adenylyl cyclases... 

There are also many neurotransmitter and hormone receptors that contribute to the fine control of cAMP formation by inhibition of adenylyl cyclase. The action of inhibitory receptors is mediated by several different forms of the Gai family, specifically the Gail, Gai2, Gai3, Gao and Goa subtypes. The Ga subunits of these isoforms can inhibit the catalytic activity of adenylyl cyclase when the enzyme is activated by either Gas or forskolin. The inhibition of catalytic activity does not occur via competition with Gas but appears to occur by an interaction at a symmetric site on the AC molecule. Gai-mediated inhibition of adenylyl cyclase is most dramatic for AC5 and AC6. A few other forms of adenylyl cyclase, most notably AC1, can be inhibited by Gao but this effect is not as potent as the inhibition of AC5 and AC6 by Gai isoforms. The GTPase activity of Gai family members can be accelerated by a large family of RGS proteins (see Chapter 19). 

Diversity of adenylyl cyclases leads to dramatic cell-to-cell differences in regulation of the cAMP pathway. The differential sensitivity of AC1-AC8 to G protein a and py subunits and to Ca2+ provides potentially complex mechanisms for the regulation of cAMP formation. The three major categories of adenylyl cyclases, based on their regulatory properties discussed above are illustrated in Figure 21-4. 

While the schemes for regulation are hypothetical, certain features do suggest different patterns of regulation of cAMP formation in cells that are consistent with the different forms of adenylyl cyclase expressed in the cell, or their phenotype. For example, in cells that contain AC1, AC3 or AC8 (Fig. 21-4A), cAMP formation would be stimulated by extracellular signals that activate receptors coupled to Gas as well as by those signals that increase Ca2+ entry into the cells. Neurons expressing AC1 may... 

The effects of Li+ upon this system have been reviewed in depth by Mork [131]. Animal studies originally demonstrated that Li+ inhibits cAMP formation catalyzed by adenylate cyclase in a dose-dependent manner [132]. The level of cAMP in the urine of manic-depressive patients changes with mental state, being abnormally elevated during the switch period between depression and mania it is proposed that Li+ s inhibitory effect upon adenylate cyclase activity may correct this abnormality. Subsequent research, in accord with the initial experiments, have shown that Li+ s interference with this second messenger system involves more than one inhibitory action. At therapeutic levels, Li+ inhibits cAMP accumulation induced by many neurotransmitters and hormones, both in... 

In humans the intronless gene encoding HR2 is located on chromosome 5. The human HR2 is a protein of 359 amino acids coupled to both adenylate cyclase and phosphoinositide second messenger systems by separate GTP-dependent mechanisms including Ga and also induces activation of c-Fos, c-Jun PKC and p70S6 kinase [16], Studies in different species and several human cells demonstrated that inhibition of characteristic features of the cells by primarily cAMP formation dominates in HR2-dependent effects of histamine. 

A. The salutary effect of p-blockers appears to be due solely to its binding to the -receptor, which prevents norepinephrine binding and stimulates cAMP formation. The other choices do not occur. 

Five subtypes of dopamine receptors have been described they are the Dj-like and Dj-like receptor groups. All have seven transmembrane domains and are G protein-coupled. The Dj-receptor increases cyclic adenosine monophosphate (cAMP) formation by stimulation of dopamine-sensitive adenylyl cyclase it is located mainly in the putamen, nucleus accumbens, and olfactory tubercle. The other member of this family is the D5-receptor, which also increases cAMP but has a 10-fold greater affinity for dopamine and is found primarily in limbic regions. The therapeutic potency of antipsychotic drugs does not correlate with their affinity for binding to the Dj-receptor. 

The primary effect of ACTH seems to be mediated via cAMP production by interaction of the hormone with differing populations of receptors. The difference between the extent of steroidogenesis and cAMP formation indicates different receptor affinities in different organs. 

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