Effectors and the regulation of second messengers

Early research into the signal transduction pathways utilized by GPCRs focussed upon pathways regulating the cellular concentration of second messenger molecules, notably cyclic AMP. Cyclic AMP regulates different cell activities through its ability to bind to specific protein kinases (cyclic AMP-dependent protein kinase (PKA)) leading to phosphorylation, and activity modification, of key protein substrates (Schramm and Selinger 1984; Francis and Corbin 1999). Such research also revealed that GPCRs regulate enzymes, which either synthesize (adenylyl cyclase/cyclic AMP) or breakdown (cyclic GMP phosphodiesterase/cyclic GMP) second messengers, via a family of heterotrimeric guanine nucleotide-binding proteins, termed G proteins (Rodbell 1980; Gilman 1987; Simon et al. 1991). Thus, these early studies led to the establishment of the 'universal' tripartite receptor-G protein-effector sequence by which GPCRs were thought to exert all of their cellular actions (Casey and Gilman 1988; Birnbaumer 1992). Although this tripartite view of GPCR signalling has pertained for many years, very recent experimental data have begun to uncover GPCR signalling pathways that appear to operate independently of G proteins (Table 5.1). A number of excellent reviews dealing with G protein-independent GPCR signalling have appeared recently (Hall etal. 1999; Heuss and Gerber 2000; Milligan and White 2001).

Many GPCRs can influence the activity of adenylyl cyclases through activation of stimulatory Gs, or inhibitory Gi proteins. The cloning of nine mammalian adenylyl cyclase isoforms,

Table 5.1 Non-ion channel effectors for GPCRs

Effector

Second messenger/mediator

G protein involvement

Second messenger-linked Adenylyl cyclase1

Cyclic nucleotide phosphodiesterase

Phospholipase C2

Phosphoinositide 3-kinase (PI3K) Phospholipase A2

Phospholipase D

Sphingosine kinase

$ cyclic AMP

I cyclic GMP I cyclic AMP

I PtdIns(4,5)P2 f PtdIns(3,4,5)P3 f PtdIns(3,4)P2 f arachidonate f lyso-PtdCho f lyso-PtdEth f phosphatidic acid f lyso-PtdOH f DAG

f sphingosine 1-phosphate

Ggust

Gßy-subunits

Gßy-subunits

Adaptor-linked

Guanine nucleotide exchange factors e.g. p115Rho-GEF Rho

Non-receptor tyrosine kinases e.g. Src/Pyk2 Btk

Other adaptor molecules e.g. p-arrestins Homers NHERF

Shc/Grb2/SOS PI3K, PLC-y

Src/JNK3

Shank/IP3R/RyR

Na+/H+-exchange g12/13

Gs, Gi/o, Gq/11. g12/13 Gßy-subunits n G protein->independent

1The mammalian adenylyl cyclase isoenzymes (AC1-AC9) are differentially regulated by Gs/G/o . Gpy-subunits. 2PLC-p isoenzymes (PLC-p1-PLCp4) exhibit different sensitivities to Gq/n and Gpy-subunits. PLC-8 isoenzymes are reported to be regulated by transglutaminase II (Gh). PLC-8 may also be regulated by Gpy-subunits.

all of which are expressed in the CNS, has allowed detailed characterizations to be undertaken (see Sunahara et al. 1996). From such work it is now clear that adenylyl cyclases are regulated not only by Gs/G;-coupled GPCRs, but also that Gq-coupled GPCRs can affect the activities of some isoenzymes through changes in the intracellular concentration of Ca2+ ([Ca2+]i) and protein kinase C activity. The profound stimulatory or inhibitory effects of changes in [Ca2+] also suggest that specific adenylyl cyclase isoenzymes represent a point of integration between GPCR and non-GPCR-mediated signalling pathways (Cooper et al. 1995; Hanoune and Defer 2001). This view is supported by the differential localization of adenylyl cyclase isoenzymes within neurons and evidence is accumulating to implicate specific isoenzymes in a variety of neuronal functions including long-term potentiation (LTP) and long-term depression (LTD) (Cooper etal. 1995; Hanoune and Defer 2001). In addition to advances in our understanding of how cyclic AMP levels are regulated within neurons, parallel advances have been made with respect to events downstream of cyclic AMP. Thus, PKA can be localized within cells through reversible interactions with anchoring proteins (Mochly-Rosen 1995), and cyclic AMP has been recognized as an important nuclear signal affecting the cell at a transcriptional level. In addition, it is now known that the actions of cyclic AMP are not universally mediated by PKA; additional cyclic AMP binding proteins (de Rooij et al. 1998; Kawasaki et al. 1998) contribute to the diversity of action of this second messenger.

Work over the past 20 years has considerably expanded the cellular metabolites considered to have second messenger (or putative second messenger) function beyond the cyclic nucleotides (Table 5.1). Thus, phospholipase Cisoenzymes (P, y, 8, and e) generate both inositol 1,4,5-trisphosphate (IP3) and sn-1,2-diacylglycerol (DAG) (Berridge 1993; Rhee 2001). The former inositol polyphosphate can mobilize Ca2+ from endoplasmic reticular stores through interaction with a receptor-operated Ca2+ channel gated by IP3 (Wilcox etal. 1998), while DAG acts through a subset of the protein kinase C isoenzymes (the cPKC and nPKCs (Tanaka and Nishizuka 1994)). The substrate for phospholipase Cs, the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), is also considered to share characteristics with classical second messengers and to regulate a number of key cell modalities (Toker 1998) including synaptic function (Osborne et al. 2001). Furthermore, PIP2 can be 3-phosphorylated by phosphoinositide 3-kinases (PI3Ks) to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3), which together with its immediate metabolite, phosphatidylinositol 3,4-bisphosphate, have demonstrable second messenger activities (Rameh and Cantley 1999; Lemmon and Ferguson 2000). In addition, a number of lipid metabolites, including long chain polyunsaturated fatty acids (e.g. arachidonic acid, generated by phospholipase A2 (Leslie 1997; Six and Dennis 2000)), phosphatidic acid (generated by phospholipase D (Liscovitch et al. 2000; Fang et al. 2001), sphingosine 1-phosphate (generated by sphin-gosine kinase (Pyne and Pyne 2000)) and other sphingolipids, have all been proposed to be intracellular messenger molecules.

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