Fluorescent guanine nucleotide analogs and G protein activation.

A.E Remmers,R Posner,R.R Neubig

doi:10.1016/s0021-9258(17)36714-5

A.E Remmers, R Posner + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(17)36714-5

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Abstract

The N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for studying G protein mechanisms. Both MANT-GTP gamma S (mGTP gamma S) and MANT-GTP (mGTP) displayed a magnesium-dependent increase in fluorescence upon binding to bovine brain G(o). A much greater increase in MANT-guanine nucleotide fluorescence was observed with excitation at 280 nm compared with 350 nm, due to energy transfer from tryptophan in G(o). G(o)-bound mGTP gamma S displays a blue-shift in its emission spectrum indicating a nonpolar environment for the G(o)-bound MANT. For the hydrolyzable analog, mGTP, the increase in fluorescence is followed by a decrease as it is hydrolyzed to mGDP. Unexpectedly, dissociation of mGDP was fast (t1/2 1.7 s) by comparison with GDP itself (t1/2 120 s). Binding of mGTP gamma S to G(o) was slow, but mastoparan increased the rate approximately 4-fold. For mGTP, mastoparan increased both the rate of binding and the peak fluorescence, even at saturating mGTP concentrations. Modeling the mGTP fluorescence kinetics in the presence and absence of mastoparan results in two novel conclusions. First, mGTP does not fully activate the G protein, even when bound. Second, mastoparan appears to increase the rate of the G protein conformational activation step, in addition to its known effect on GDP release.

Highlights

The N-methyl-3‘-0-anthranoy( Ml A N T ) guanine nucle- ers consisting of a guanine nucleotide binding subunit (Ga, otide analogs are useful environmentallsyensitive fluo- 39-55 kDa) andtwo additional subunits (Py), or as low molecurescent probes for studyinGgprotein mechanisms
MGTP, the changes in transducin binding t o rhodopsin (3).More recently, increase in fluorescence is followed by a decreaases it is G proteinintrinsic fluorescence was usedt o monitor transducin hydrolyzed to mGDP
Activated G protein interacts with effector proteins such aasdenylyl cyclase, phospholipases, ionchannels, or cGMP phosphodiesterase to modulate intracellular second messenger pathways.Signal transducing GTP-binding proteins aregenerally classified as either high molecular weight GTP-binding proteins which are heterotrimsolutions (8, 10, 11).the receptor-stimulated G protein activation-deactivation cycle has not been studied, most likely due to thehigh background and relatively small signals available from intrinsic tryptophan fluorescence

Summary

G PCrootneifnormation

The plateau fluorescence decreased rapidly upon addition of 10 p~ GTPyS (Fig.[3] A ) indicating thateven mGDP where k, and k , are the rate constants for association and dissociation of GDP,mGTP, and mGDP for n = 1,2,and 4,respectively. MGTP is hydrolyzed with a half-time of 6.0 2 0.5 min, and mGDP appears with a nearly identical half-time of 7.5 2 0.8 min (Fig. 3B).The complete conversion of mGTP to mGDP RelativQe uantum Eelds of Go-bound MANTGuanine Nucleotides-To show that MANT-guanine nucleotide fluorescence is due to saturable bindingGtoprotein and to assess the relative quantum yields of Go-bound nucleotides, several con-

RESULTS

DISCUSSION

Findings

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