Functional effector molecules

Many biochemical and biophysical studies of CAP-DNA complexes in solution have demonstrated that CAP induces a sharp bend in DNA upon binding. This was confirmed when the group of Thomas Steitz at Yale University determined the crystal structure of cyclic AMP-DNA complex to 3 A resolution. The CAP molecule comprises two identical polypeptide chains of 209 amino acid residues (Figure 8.24). Each chain is folded into two domains that have separate functions (Figure 8.24b). The larger N-terminal domain binds the allosteric effector molecule, cyclic AMP, and provides all the subunit interactions that form the dimer. The C-terminal domain contains the helix-tum-helix motif that binds DNA. 

The Gpy dimer appears to function as a rigid unit with critical residues positioned to interact with Ga-GDP. Whereas Ga activation proceeds through nucleotide-dependent structural reorganization, activation of Gpy occurs solely as a function of its release from Ga. As we will see, the Ga subunit acts as a negative regulator of Gpy by masking sites on the surface of Gpy that interact with downstream effector molecules. 

Ca2+ is the main intracellular signalling molecule in smooth muscle. Fluctuation in local cytoplasmic [Ca2+] near Ca2+-sensitive effector molecules allows for specific regulation of multiple functions. These temporal fluctuations and spatial variations of cytoplasmic [Ca2+] are dependent on the interactions of ion transport proteins located in the plasma membrane (PM) and membranes of the sacoplasmic reticulum (SR), nuclear envelope and mitochondria. These... 

In addition to GnRH, several other effector molecules modulate secretion of gonadotrophins. These include not only inhibins and activins, but also gonadal steroids. All of these combine via complex feedback loops to modulate gonadotrophin production and, hence, reproductive function (Figure 8.15). 

Metal ions can serve as effector molecules as well as control the DNA-binding activity of regulatory proteins. An example is the regulation of the metallothionein gene in eucaryotes (Fig. 1.23). The metallothioneins are small, cysteine rich proteins which can specifically bind metal ions like Cu or Zn The complexation of metal ions functions to sequester the ions in a form that is not damaging to the cell. 

The regulation of the activity of enzymes by the binding of effector molecules is a ubiquitous and general principle for the fine timing and control of metabolic activity. Effector molecules are often low molecular weight organic compounds. Proteins and metal ions can also exercise the function of effectors. The effector molecules bind specifically to the enzymes and the binding results in inhibition or stimulation of enzymatic activity. 

The Ras protein has low intrinsic GTPase activity. This may be increased ca. 10 -fold by the corresponding GTPase-activating protein (see also Chapter 9). In comparison, the intrinsic rate of GTP hydrolysis of transducin is ca. 100-fold higher that that of the Ras protein. The effector molecule next in the reaction chain, the cGMP phosphodiesterase, functions as the GAP here and stimulates GTPase activity of the transducin 100-fold. 

Fig. 5.16. Functional cycle of the heterotrimeric G-proteins. a) The G-proteins exist in the ground state as a heterotrimeric complex (G GDP) (Py)- b) The activated receptor binds to the inactive heterotrimeric complex of the G-protein and leads to dissociation of the bound GDP and the Pyeomplex. c) Binding of GTP to the empty G -subunit transforms the latter into the active G GTP state. G GTP interacts with an effector molecule in the sequence El and activates the latter for further signal transmission. The released Py-complex may also take part in signal conduction by binding to a corresponding effector molecule E2 and activating the latter for further signal conduction, d) Hydrolysis of the bound GTP terminates the signal transduction via the a-subunit.

The interaction of G GTP with the corresponding effector molecule leads to inactivation of the former and thus to initiation of the next step in the signal transmission chain. The Pycomplex released during activation can also perform a signal-mediating function (see 5.5.7). 

The rate of GTP hydrolysis may also be increased via the downstream effector molecule. Phospholipase C-pi stimulates the intrinsic GTPase activity of the corresponding Gq-ii by close to two orders of magnitude (Bernstein et al., 1992). A further effector molecule, adenylyl cylase, has been shown to function as a GAP for the monomeric G -GTP state (Scholich et al., 1999). 

The Py-complex does not show any great structural differences in the free and G -boimd forms. Activation of the Py-complex for the interaction with the corresponding effector molecule (see below) appears to be based only on its release from the inactive Ga GDP Py complex. The Ga-subimit has the function of a negative regulator here, that inactivates the PY-compIex by masking the interaction region for signal proteins next in the sequence. 

Originally, it was assumed that the Py-complex only played a passive role in the functional cycle of the G-proteins. It soon became apparent, however, that the Py-complex, in addition to binding to the a-subimit, also carries out other functions and interacts specifically with corresponding effector molecules (review article Neer, 1995). Tlie Py-complex must be assigned its own regulatory function, it takes part itself in the propagation and termination of signal transmission. 

The products of the PI3-kinase reaction are different phosphoinositide derivatives phosphorylated at the 3 position, of which PtdIns(3,4,5)P3 has the greatest regulatory importance. PtIns(3,4,5)P3, like cAMP, has the function of a messenger substance that activates effector molecules in the sequence for further signal conduction. In contrast to cAMP, Ptdlns(3,4,5)P3 is localized in the cell membrane and performs its function in close association with processes at the cell membrane. 

Autophosphorylation may be attributed two functions first, activation of the own Tyr kinase activity by cancelling autoinhibition second, creation of binding sites for corresponding effector proteins, in that Tyr phosphate binds to the SH2 domains or PTB domains (see 8.2) of effector molecules (Fig. 8.6). 

Permanent or transient association with subcellular structures, and variable subcellular distribution, are characteristic for the cytoplasmic tyrosine kinases. Tire nonreceptor tyrosine kinases are intracellular effector molecules that can associate with specific substrates during the process of signal transduction and activate these by tyrosine phosphorylation, to pass on the signal. Many of the functions of the nonreceptor tyrosine kinases are performed in the iimnediate vicinity of the cell membrane, whether a signal is received from an activated membrane receptor or a signal is passed on to a membrane-associated protein. 

Fig. 9.12. Overview of the Ras signaling pathway. Signals from at least three major signaling pathways meet at the Ras protein. Activation of the Ras protein may be initiated by receptor tyrosine kinases, by G-protein-coupled receptors and by receptors with associated tyrosine kinases. The nature of the communication between the Ras protein and receptors with associated tyrosine kinase or G-protein-coupled receptors is mostly unknown. From the activated Ras protein, the signal is passed to various effector molecules including members of the MEK kinases, PI3-kinase, pl20 GAP and Ral-GEFs. The best understood is the effector function of Raf kinase, which passes a signal to the transcription level via the MAP kinase pathway.

Antigen MHC class I complexes have a different function and present their antigen to CD8+ cytolytic T cells, which produce an appropriate group of effector molecules leading to apoptosis of target cells. 

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