GTP-Binding Protein alpha Subunits

About: GTP-Binding Protein alpha Subunits is a research topic. Over the lifetime, 304 publications have been published within this topic receiving 19915 citations.

Obligatory role in GTP hydrolysis for the amide carbonyl oxygen of the Mg2+-coordinating Thr of regulatory GTPases

Abstract: When G-protein α subunits binds GTP and Mg2+, they transition from their inactive to their active conformation. This transition is accompanied by completion of the coordination shell of Mg2+ with electrons from six oxygens: two water molecules, the s and γ phosphoryls of GTP, a helix-α1 Ser, and a switch I domain (SWI) Thr, and the repositioning of SWI and SWII domains. SWII binds and regulates effector enzymes and facilitates GTP hydrolysis by repositioning the γ-carbonyl of a Gln. Mutating the Ser generates regulatory GTPases that cannot lock Mg2+ into its place and are locked in their inactive state with dominant negative properties. Curiously, mutating the Thr appears to reduce GTP hydrolysis. The reason for this difference is not known because it is also not known why removal of the Thr should affect the overall GTPase cycle differently than removal of the Ser. Working with recombinant Gsα, we report that mutating its SWI-Thr to either Ala, Glu, Gln, or Asp results not only in diminished GTPase activity but also in spontaneous activation of the SWII domain. Upon close examination of existing α subunit crystals, we noted the oxygen of the backbone carbonyl of SWI-Thr and of the γ-carbonyl of SWII Gln to be roughly equidistant from the oxygen of the hydrolytic H2O. Our observations indicate that the Gln and Thr carbonyls play equihierarchical roles in the GTPase process and provide the mechanism that explains why mutating the Thr mimics mutating the Gln and not that of the Ser.

Competition for Gβγ dimers mediates a specific cross-talk between stimulatory and inhibitory G protein α subunits of the adenylyl cyclase in cardiomyocytes

Abstract: Heterotrimeric G proteins are key regulators of signaling pathways in mammalian cells. Beyond G protein-coupled receptors, the amount and mutual ratio of specific G protein α, β, and γ subunits determine the G protein signaling. However, little is known about mechanisms that regulate the concentration and composition of G protein subunits at the plasma membrane. Here, we show a novel cross-talk between stimulatory and inhibitory G protein α subunits (Gα) that is mediated by G protein βγ dimers and controls the abundance of specific Gα subunits at the plasma membrane. Firstly, we observed in heart tissue from constitutively Gαi2- and Gαi3-deficient mice that the loss of Gαi2 and Gαi3 was accompanied by a slight increase in the protein content of the nontargeted Gαi isoform. Therefore, we analyzed whether overexpression of selected Gα subunits conversely impairs endogenous G protein α and β subunit levels in cardiomyocytes. Integration of overexpressed Gαi2 subunits into heterotrimeric G proteins was verified by co-immunoprecipitation. Adenoviral expression of increasing amounts of Gαi2 led to a reduction of Gαi3 (up to 90 %) and Gαs (up to 75 %) protein levels. Likewise, increasing amounts of adenovirally expressed Gαs resulted in a linear 75 % decrease in both Gαi2 and Gαi3 protein levels. In contrast, overexpression of either Gαi or Gαs isoform did not influence the amount of Gαo and Gαq, both of which are not involved in the regulation of adenylyl cyclase activity. The mRNA expression of the disappearing endogenous Gα subunits was not affected, indicating a posttranslational mechanism. Interestingly, the amount of endogenous G protein βγ dimers was not altered by any Gα overexpression. However, the increase of Gβγ level by adenoviral expression prevented the loss of endogenous Gαs and Gαi3 in Gαi2 overexpressing cardiomyocytes. Thus, our results provide evidence for a novel mechanism cross-regulating adenylyl cyclase-modulating Gαi isoforms and Gαs proteins. The Gα subunits apparently compete for a limited amount of Gβγ dimers, which are required for G protein heterotrimer formation at the plasma membrane.

ADP-ribosylation of alpha i3C20 by the S1 subunit and deletion peptides of S1 of pertussis toxin.

Abstract: Recombinant S1 subunit of PT (rS1) and two carboxyl-terminal deletion peptides, C180 and C204, which comprise the amino-terminal 180 and 204 amino acids of S1, respectively, were analyzed for the ability to ADP-ribosylate alpha i3C20, a synthetic peptide composed of the 20 carboxyl-terminal amino acids of the alpha subunit of the heterotrimeric G protein Gi3. Under linear velocity conditions, C180 ADP-ribosylated alpha i3C20 at a 3-fold higher rate than either C204 or rS1. At variable NAD, rS1, C204, and C180 ADP-ribosylated alpha i3C20 with similar initial velocities which followed Michaelis-Menten kinetics. In contrast, at variable alpha i3C20, rS1, C204, and C180 ADP-ribosylated alpha i3C20 with different initial velocities. At variable alpha i3C20, C204- and rS1-catalyzed ADP-ribosylation followed Michaelis-Menten kinetics, while the velocity curve generated by C180 diverged from Michaelis-Menten kinetics. The rates of initial velocity of C180 did not fit the Lineweaver-Burk equation, but could be transformed into the Hill equation which yielded a Hill coefficient of 2. This predicted that C180 possessed cooperativity between the two substrate binding sites. Other experiments showed that C180 ADP-ribosylated alpha i3C20 at 60% of the rate for the ADP-ribosylation of Gt. These data showed that the entire catalytic mechanism for ADP-ribosylation resides within the first 180 amino acids of S1 and that the carboxyl-terminal 55 residues of S1 allow the ADP-ribosylation of alpha i3C20 to proceed via Michaelis-Menten kinetics. These data along with earlier studies (Krueger & Barbieri, 1993) were also consistent with the presence of two Gt protein binding sites within S1.(ABSTRACT TRUNCATED AT 250 WORDS)