Summary
The concept that a single G protein-coupled receptor (GPCR) can sequentially activate a number of heterotrimeric G proteins on the floor of the plasma membranes derived from 2 separate experimental arguments made previous to 1990, and is now typically accepted. GPCRs catalyze GDP/GTP trade to advertise G protein activation, and the definition of a catalyst calls for turnover of a number of substrate molecules. Massively catalytic G protein activation is finest demonstrated within the mammalian photoreceptor membrane, the place nearly all of the membrane protein is the GPCR rhodopsin and the overwhelming majority of GTP binding websites is the Gt (see essays by Arshavsky, Liebman, …). In different cells, the place each GPCR and its G protein targets could also be nicely under 0.1% of plasma membrane protein, catalytic turnover by GPCRs stays onerous to quantitate. In what membranes it happens, which receptors and G proteins do it, if and the way it’s regulated, and which protein(s) is (are) the diffusing species are all variables.
The primary suggestion that GPCRs and/or G proteins diffuse laterally and {that a} receptor can activate a number of G proteins got here from the work of Levitzki and coworkers,1-3 who used adenylyl cyclase exercise as a surrogate measure of Gs activation; Gs was itself found at about the identical time. They confirmed that covalent inactivation of β-adrenergic receptors in erythrocyte membranes progressively decreased the obvious first-order fee of activation of adenylyl cyclase by non-hydrolyzable GTP analogs at a set agonist focus with out lowering maximal stimulation. Such inactivation additionally shifted the EC50 for agonist to larger concentrations. The variety of catalytic turnovers for every receptor earlier than cyclase was absolutely activated could possibly be estimated from these information to be about 10. With the work of Selinger and coworkers,4-7 who confirmed that β-adrenergic agonists promote the GDP/GTP trade step within the receptor-stimulated GTPase cycle, the thought of trade catalysis by receptors entered the evaluate literature by 1980,8 if not earlier. Even at this level, nevertheless, it was additionally clear that not all receptors recycle freely. Braun and Levitzki9 confirmed that an adenosine receptor appeared by the above standards to be tightly coupled to Gs, with no signal of multi-molecular turnover.
Correct quantitation of catalytic turnover of G proteins by GPCRs is troublesome in intact cells or remoted plasma membranes, however turned potential when the purified proteins had been reconstituted into phospholipid vesicles. In an early effort, Pedersen and Ross10 discovered {that a} β-adrenergic receptor may catalyze nucleotide trade on about 6 Gs molecules, and Asano et al.,11 utilizing extra correct quantitation, demonstrated a minimum of 8 turnovers. A kinetic evaluation of Asano’s information just like that carried out by Levitzki and coworkers argued {that a} single receptor may flip over about 50 Gs molecules. Utilizing a reconstituted system just like that of Asano et al., Cerione et al.12 additionally discovered that the α2-adrenergic receptor may catalyze GDP/GTP trade on about 7 molecules of Gi. Mirroring Braun, Senogles et al.13 then discovered that the reconstituted D2 dopamine receptor didn’t flip over a number of Gi molecules. Some receptors do; some don’t. It’s unsure whether or not receptors that do or don’t regulate a number of G protein molecules in phospholipid vesicles may behave in another way in cells, though some co-fractionation research have argued that these behaviors are constant.
Given {that a} receptor can sequentially catalyze GDP/GTP trade on a number of G proteins, a minimum of in some instances, the extra related physiologic query is whether or not it does so inside the lifetime of the G protein’s GTP-activated state. If it may possibly, then a single agonist-liganded receptor can keep the activation of a number of G proteins at steady-state, resulting in molecular amplification of the sign. Catalytic amplification can even enhance agonist efficiency (lower EC50) as a result of fewer agonist-bound receptors can activate the identical variety of G proteins.
Even with catalytic turnover, although, amplification will solely happen if the speed of receptor-catalyzed GDP/GTP trade is quicker than the speed of hydrolysis of G protein-bound GTP. Fee constants for G protein deactivation, with or with out stimulation by GTPase-activating proteins (GAPs), haven’t been measured instantly in dwelling cells or in intact membranes apart from the photoreceptor disc. The deactivation fee fixed for Gs and Gi in cells is estimated to be ~0.05–0.1 s−1, primarily based on the charges of sign termination,14,15 and the charges of hydrolysis of GTP sure to purified Gαi and Gαs, are just like this worth. Different G proteins are slower: ~0.02 s−1 for Gαq16 and ~0.002 s−1 for Gαz,17 Gα12 and Gα13.18 GAPs can enhance these charges as a lot as 2000-fold.19,20 Considerably sooner charges of G protein activation and deactivation in cells have been estimated in accordance with effector activation/deactivation charges (above) or with fluorescent G protein activation biosensors.21,22 Whereas they supply a phenomenal perception into the time course of signaling, on/off kinetics has not clarified the amplification query. To do that would require stoichiometric info that’s not obtainable from ensemble fluorescence information. Generally, stoichiometric relationships among the many protein elements of plasma membrane signaling networks are poorly understood. They’re central to problems with molecular amplification, and so they additionally dictate whether or not these networks act as first-order (linear) or non-first-order (nonlinear) transducers.
Past the not-so-simple activation/deactivation kinetic points are the questions of mobile constraints on receptor and G protein diffusion. Clearly some receptors and G proteins are extremely constrained of their movement. Constraints embody secure binding to scaffolding proteins on the molecular scale (1–2 nm) and membrane area restrictions on the 100–1000 nm scale. Different mechanistic constraints may additionally come up. For instance, we have now proposed that a method during which G protein modules use GAPs to speed up response charges is to advertise long-term (10 s vs 0.1 s) receptor-G protein binding.20,23 This mechanism overcomes inhibition by the GAP, however at the price of molecular amplification.
If catalytic amplification happens on the preliminary receptor-G protein step within the signaling pathway, we have now to know absolutely the fee constants for precise nucleotide trade and GTP hydrolysis, and we don’t for any system I do know of. One can envision double-label, single-molecule approaches to the issue regardless that the expertise is just not now obtainable. I predict that the query of molecular amplification in cells will depend upon such optical methods perfected to present us each kinetic and stoichiometric information, in addition to molecular-scale location. Imaging expertise is pushing towards this objective.
Disclosure of Potential Conflicts of Curiosity
References
Codecs: – “g protein coupled receptors steps”
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“g protein coupled receptors steps”