Transduction systemsG proteins

It was at first believed that the enzymes responsible for formation of second messengers were directly linked to the membrane-bound receptors. However, the pioneering work of Gilman and of Rodbell,(6) for which they were awarded the Nobel Prize in 1994, showed that another class of proteins exist which mediate between the receptors and effector enzymes, and are thus responsible for the phenomenon now known as 'receptor-effector coupling'. Activation of these proteins requires binding of the guanine nucleotide guanosine triphosphate, and the proteins, initially referred to as N (nucleotide binding) proteins, are now universally known as G proteins (guanyl nucleotide binding proteins).

Currently four major families of G proteins are known to exist (Table 1), defined on the basis of their sequence homologies. (I) It was initially thought that each G

protein coupled to a single effector molecule only, and the G proteins were thus designated by letters specifying this action, such as G s and Gi for stimulation and inhibition of adenylate cyclase respectively. It is now recognized that G proteins are 'promiscuous' in that they can interact with more than one effector system. G s, for example, is capable not only of stimulating adenylate cyclase but also of opening L-type calcium channels. G o, a member of the Gi class, is linked to calcium channels and in some systems to hydrolysis of the polyphosphoinositides via stimulation of phospholipase C. Another member of the G i class is the protein Gt, originally named transducin, which is coupled to the enzyme cyclic guanosine monophosphate (cGMP) phosphodiesterase. The two remaining classes of G proteins comprise Gq, which is coupled to phospholipase C-b, and G12, for which the effector system is not known. Each of the G proteins consists of a-, b-, and g-subunits, with the a-units conferring specificity. The bg-units were originally thought to be similar or interchangeable for all G-protein heterotrimers. However, at least five b-subunits and eleven g-subunits are now known to exist. It is not known whether certain a-subunits prefer certain bg-complexes. Molecular cloning studies have identified at least four forms of the a-subunit of Gs, which may result from alternative splicing of a single precursor mRNA. Three forms of the a-subunit of G i, which appear to be products of three separate genes, have been identified and named Gi1-a, Gi2-a, and Gi3-a. Neural tissue contains predominantly Gi1-a and Gi2-a, while platelets contain Gi2-a and Gi3-a.

Table 1 Mammalian G-protein a-subunits

The mechanism by which G proteins are activated is now understood ( Fig 3). Interaction of a ligand with a receptor results in dissociation of the a-subunit from the complex and exchange of the bound guanosine diphosphate for guanosine triphosphate. The dissociated a-units then activate the relevant intracellular effectors and also hydrolyse the bound guanosine triphosphate to guanosine diphosphate via an intrinsic guanosine triphosphatase, thus facilitating reassociation of the inactive a-b-g heterotrimer and terminating the signal. Adenosine diphosphate ribosylation of the a-units by bacterial toxins accounts both for the diseases produced by the relevant bacteria and also enables intervention in the G-protein activation cycle. This can be used to establish the identity of the G protein involved in a particular physiological process. Cholera toxin modification of Gs inhibits guanosine triphosphatase activity and leads to constitutive stimulation of cAMP formation, while pertussis toxin modifies Gi and Go, and blocks signal transduction by uncoupling the G protein from its receptor. Sensitivity to pertussis toxin is indeed a characteristic of the Gi group of G proteins, the one exception being Gz. It is important to note that receptors may be coupled to phosphoinositide hydrolysis by pertussis-toxin-sensitive Gi or Go, or by pertussis-toxin-insensitive Gq.

Selenium Nodes

Fig. 3 The G-protein cycle of activation. During activation by a receptor (R), guanosine triphosphate (GTP) binds to the a-unit of the G-protein heterotrimer (a-b-g) and induces its dissociation into a-GTP and bg units. GTP is then hydrolysed into guanosine diphosphate (GDP) by a guanosine triphosphatase (GTPase) enzyme intrinsic to the a-unit, and the inactive a-GDP unit then recombines with bg. (Reproduced with permission from P.C. Sternweis and I.-H. Pang (1990). The G protein-channel connection. Trends in Neurosciences, 13, 122-6.) Printed with permission from Elsevier Science.

Understanding of the roles played by the different subunits of G proteins has undergone a drastic transition in recent years. It was initially believed that messages were carried by the a-units only, and that the bg-units served to terminate these messages by combining with and thus inactivating the a-units. bg-units are now known to have well-defined effects on some isoforms of the classical second-messenger systems, including phospholipases C-b 2 and C-b3 and several G-protein-responsive potassium, calcium, and perhaps also sodium ion channels. In addition, some isoforms of adenylate cyclase are activated by bg, while others are inhibited (Table. ...2). While in vitro studies have shown that many combinations of a- and bg-units can exist, in vivo a high degree of specificity may exist both for interactions between a- and bg-units and for interactions between receptors and G proteins. This has been demonstrated using antisense oligonucleotides to suppress translation of specific G-protein subunits. Examination of subsequent impaired cellular responses indicates whether the specific subunit is involved in the signal transduction processes leading to the response. For example, it was shown that inhibition of calcium channels by somatostatin receptors in GH3 cells is mediated by the combination ao2b1g3, while inhibition by M4 muscarinic receptors is mediated by ao1b1g4.(8.)

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Table 2 Mammalian adenylate cyclases (grouped by structural relatedness)

The central role played by G proteins in the signal transduction process is illustrated by the fact that G-protein molecules vastly outnumber the receptor molecules in any given cell. G proteins thus act to amplify the relatively small chemical signals generated by transmitter and receptor molecules. The high degree of complexity generated by the interactions of G-protein-coupled receptors with G proteins may be one mechanism whereby neurons acquire the flexibility necessary to generate the wide variety of responses observed in the central nervous system.

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