The a subunits

Purification of T from retinal rod outer segments and of G0 from brain, provided yields of these proteins that were sufficient for partial amino acid sequence analysis of their proteolytic fragments. This analysis revealed that a subunits of G proteins, while quite distinct from each other in general terms, are nevertheless similar. A partial sequence of 21 amino acids was determined to be common in bovine rod cell T and bovine brain G0 [168], On the basis of this sequence, and other amino acid sequence analysis, four laboratories cloned cDNAs coding for transducin. The deduced amino acid structure of three of the cDNAs is the same [169-171] the fourth differed [172]. Peptide-directed antibodies designed to distinguish between the two cloned forms localized one to rod cells (T-r) and the other to cone cells (T-c) [173], 

At this time, the nucleotide sequences of the cDNAs encoding nine distinct a subunits have been published T-r [169-171] T-c [172] Gs-la, Gs-lb, Gs-2a and Gs-2b [12-17] Gj-1 [10] and Gr2 [14-16] and G0 [14]. All have as identifying signature a common 18 amino acid identity box flanked on both sides by either Arg or Lys  

Signal transducing proteins general subunit structure 

G Found in (mam- Subunit composition Properties of subunits Function of protein Regulated by Effect of HR corn- 

Mr = 39000 binds GTP hydrolyzes GTP ADP-ribosylated by cholera and pertussis toxins 

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The Fe-N mode is at 222 in the R state and 207 cnY in the T state for the a subunits, but only shifted to 218 T state for the (3 subunits. This is consistent with the interpretation that the Fe-imidazole interations are weakened more in the T state of the a subunits than p subunits. Time-resolved resonance Raman studies have shown that the R T switch is complete on a 10 ps tuuescale [38]. Finally, UV excitation of the aromatic protein side chains yields... 

Figure B2.1.7 Transient hole-burned speetra obtained at room temperature with a tetrapyrrole-eontaining light-harvesting protein subunit, the a subunit of C-phyeoeyanin. Top fluoreseenee and absorption speetra of the sample superimposed with die speetnuu of the 80 fs pump pulses used in the experiment, whieh were obtained from an amplified CPM dye laser operating at 620 mn. Bottom absorption-diflferenee speetra obtained at a series of probe time delays.

Figure B2.1.10 Stimulated photon-echo peak-shift (3PEPS) signals. Top pulse sequence and iuterpulse delays t and T. Bottom echo signals scaimed as a fiinction of delay t at tluee different population periods T, obtained with samples of a tetrapyrrole-containing light-harvesting protein subunit, the a subunit of C-phycocyanin.

Pseudohypoparathyroidism is characterized by end-organ resistance to parathyroid hormone (98,108). This disease takes various forms, including Albright s hereditary osteodystrophy, which has unusual physical features and a generalized resistance to G-protein-linked hormones that function through cAMP as a second messenger. This defect is associated with a deficiency in the levels of the a-subunit of (109). Because this defect may be generalized, such patients also have olfactory dysfunction (110). 

Lambright, D.G., et al. Structural determinants for activation of the a-subunit of heterotrimeric G protein. Nature 369 621-628, 1994. 

In the T = 4 structure there are 240 subunits (4 x 60) in four different environments, A, B, C, and D, in the asymmetric unit. The A subunits interact around the fivefold axes, and the D subunits around the threefold axes (Figure 16.7). The B and C subunits are arranged so that two copies of each interact around the twofold axes in addition to two D subunits. For a T = 4 structure the twofold axes thus form pseudosixfold axes. The A, B, and C subunits interact around pseudothreefold axes clustered around the fivefold axes. There are 60 such pseudothreefold axes. The T = 4 structure therefore has a total of 80 threefold axes 20 with strict icosahedral symmetry and 60 with pseudosymmetry. 

The asymmetric unit contains one copy each of the subunits VPl, VP2, VP3, and VP4. VP4 is buried inside the shell and does not reach the surface. The arrangement of VPl, VP2, and VP3 on the surface of the capsid is shown in Figure 16.12a. These three different polypeptide chains build up the virus shell in a way that is analogous to that of the three different conformations A, C, and B of the same polypeptide chain in tomato bushy stunt virus. The viral coat assembles from 12 compact aggregates, or pen tamers, which contain five of each of the coat proteins. The contours of the outward-facing surfaces of the subunits give to each pentamer the shape of a molecular mountain the VPl subunits, which correspond to the A subunits in T = 3 plant viruses, cluster at the peak of the mountain VP2 and VP3 alternate around the foot and VP4 provides the foundation. The amino termini of the five VP3 subunits of the pentamer intertwine around the fivefold axis in the interior of the virion to form a p stmcture that stabilizes the pentamer and in addition interacts with VP4. 

Indole, the product of the a-reaction and the reactant for the /3-reaction, is passed directly from the a-subunit to the /3-subunit and cannot be detected as a free intermediate. 

Myristic acid may be linked via an amide bond to the a-amino group of the N-terminal glycine residue of selected proteins (Figure 9.18). The reaction is referred to as A -myristoylation and is catalyzed by myristoyl—CoAtprolein N-myris-toyltransferase, known simply as NMT. A -Myristoyl-anchored proteins include the catalytic subunit of cAMP-dependent protein kinase, the ppSff tyrosine kinase, the phosphatase known as calcineurin B, the a-subunit of G proteins (involved in GTP-dependent transmembrane signaling events), and the gag proteins of certain retroviruses, including the FHV-l virus that causes AIDS. 

The hormonal stimulation of adenylyl cyclase is effected by a transmembrane signaling pathway consisting of three components, all membrane-associated. Binding of hormone to the external surface of a hormone receptor causes a conformational change in this transmembrane protein, which in turn stimulates a GTP-binding protein (abbreviated G protein). G proteins are heterotrimeric proteins consisting of a- (45-47 kD), /3- (35 kD), and y- (7-9 kD) subunits. The a-subunit binds GDP or GTP and has an intrinsic, slow... 

FIGURE 2.6 Production of cyclic AMP from ATP by the enzyme adenylate cyclase. Cyclic AMP is a ubiquitous second messenger in cells activating numerous cellular pathways. The adenylate cyclase is activated by the a subunit of Gs-protein and inhibited by the a-subunit of Gj-protein. Cyclic AMP is degraded by phosphodiesterases in the cell. 

FIGURE 2.7 Production of second messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) through activation of the enzyme phospholipase C. This enzyme is activated by the a- subunit of Gq-protein and also by Py subunits of Gj-protein. IP3 stimulates the release of Ca2+ from intracellular stores while DAG is a potent activator of protein kinase C. 

Cohn, D. H., et al. (1985). Nucleotide sequence of the luxA gene of Vibrio harveyi and the complete amino acid sequence of the a subunit of bacterial luciferase. J. Biol. Chem. 260 6139-6146. 

The a subunits, for which two isoforms exist in mammals (al, a2), contain conventional protein serine/threonine kinase domains at the N-terminus, with a threonine residue in the activation loop (Thr-172) that must be phosphorylated by upstream kinases (see below) before the kinase is active. The kinase domain is followed by an autoinhibitory domain, whose effect is somehow relieved by interaction with the other subunits. The C-terminal domain of the a subunit is required for the formation of a complex with the C-terminal domain of the (3 subunit, which in turn mediates binding to the y subunit. The al and a2 catalytic subunit isoforms are widely distributed, although a2 is most abundant in muscle and may be absent in cells of the endothelial/hemopoietic lineage. 

Protein toxins acting intracellularly are often composed of two subunits (A/B model). One subunit is catalytic (A-subunit) and the other is responsible for binding and cell entry (B-subunit). Following binding to an extracellular membrane receptor, the toxins are endocytosed. From the endosomes, the A-subunit is directly (pH dqDendent) transferred into the cytosol (e.g., diphtheria toxin and anthrax toxin) or the toxin is transported in a retrograde manner via the golgi to the ER (e.g., cholera toxin), where translocation into the cytosol occurs [1]. 

Cholera toxin is a protein toxin of Vibrio choleme. Toxin ADP-ribosylates the a-subunit of the Gs heterotrimeric... 

The insulin-binding domain of the INSR is located within a cystein-rich region of the a-subunits. Alternative splicing of exon 11 generates two isoforms of the a-subunit which differ in their C-terminus and in their tissue distribution (type A leukocytes type B liver type A and B skeletal muscle and fat). The isoforms differ in their affinity to insulin (A > B), but then-relevance for normal and impaired insulin action is not entirely clear [1,2]. 

Insulin Receptor. Figure 1 Structure and function of the insulin receptor. Binding of insulin to the a-subunits (yellow) leads to activation of the intracellular tyrosine kinase ((3-subunit) by autophosphorylation. The insulin receptor substrates (IRS) bind via a phospho-tyrosine binding domain to phosphorylated tyrosine residues in the juxtamembrane domain of the (3-subunit. The receptor tyrosine kinase then phosphorylates specific tyrosine motifs (YMxM) within the IRS. These tyrosine phosphorylated motifs serve as docking sites for some adaptor proteins with SRC homology 2 (SH2) domains like the regulatory subunit of PI 3-kinase. 

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