The G proteins identified by function and purification

The first evidences for a stimulatory role of GTP in regulation of adenylyl cyclase systems were published in 1971 [1,2]. By 1980 a separate component, responsible for mediation of hormonal stimulation of adenylyl cyclases, had been purified [3]. This component, initially referred to as G/F and Ns, is now called Gs. It is a het-erotrimeric complex composed of a subunits that migrate on SDS-PAGE at 42 and 52 kDa [3], (3 subunits of ca. 35 kDa [3], and y subunits of 6-10 kDa [4] (For reviews see Refs. 5 and 6). Its a subunits are ADP-ribosylated by CTX [7], dissociate from the holocomplex after activation [8,9] and hydrolyze GTP [10]. The a subunits have been cloned in several laboratories [11-17] and their amino acid composition has been deduced from the cDNA nucleotide sequence. The amino acid sequence of one of two types of /3 subunits, called /336 [18], has also been deduced from its cDNA [19-21]. The amino acid sequence of the y subunit is not yet known. 

Gs is established to be the stimulatory regulatory component of adenylyl cyclase. This was first demonstrated by its ability to reconstitute the adenylyl cyclase system of cyc cells [3,22] concomitant with the reappearance of CTX labeling [23]. Cyc cells are derived from S49 murine lymphoma cells and lack Gs as indicated by lack of stimulation of adenylyl cyclase by NaF, GTP analogs and hormones (in spite of the presence of stimulatory receptors), by lack of substrate for CTX and by lack of mRNA encoding Gs-a subunits. Moreover, pure Gs also stimulates a resolved C preparation [24], as well as fully purified C [25,26] of adenylyl cyclase, both in solution [27] and after reconstitution into phospholipid vesicles [28]. Thus, by all criteria the purified Gs is functional Gs. 

The activation of Gs has been studied extensively both in native membranes and with purified Gs in solution. Non-hydrolyzable GTP analogs, but not GTP, activate soluble Gs. However, both the analogs and GTP elicit Gs activation in membranes, 

Pure Gs that has been incorporated into phospholipid vesicles exhibits a very low GTPase activity, ranging from 0.02 to 0.05 mol hydrolyzed per min per mol of Gs [31], Co-incorporation into these vesicles of pure beta-adrenergic receptors increases this activity by a factor of 2-3 to 0.05-0.1 mol of GTP hydrolyzed per min per mol of Gs. Stimulation of the receptor with a beta-adrenergic agonist (isoproterenol) results in a further increase in GTP hydrolysis to rates of ca 1.0 mol of GTP hydrolyzed per min per mol of Gs [31,32], 

The a and 13 subunits of Gs are water soluble the y subunits, on the other hand are strongly hydrophobic. Since the a/3y complex is hydrophobic, it is currently thought that Gs is a peripheral membrane protein anchored into the-inner leaflet of the membrane bilayer through its y subunit. The possibility exists that, upon activation, a GTP complexes could be released from the membrane. This led Rodbell to postulate functions for such programmable second messengers [33]. 

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