Native receptor

Non-NMDA ionotropic glutamate receptors (the majority sodium channel containing) can be subdivided into a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) (comprising cloned subunits GluRl ) and kainate (GluR5-7, KAl-2) preferring receptors, with native receptors most likely to comprise either homo- or heteromeric pentamers of these subunits. 

Expression studies in Xenopus oocytes or transfected cell lines originally suggested that functional GABA-activated chloride channels could be formed by receptor subunits of each class in isolation. However, much better expression occurs with two or more subunit types in combination and it is likely that most native receptors contain at least three different subunits. Co-expression of a and /I subunits results in the assembly of... 

Given that the pharmacological and biophysical properties of recombinant GABAa receptors have been shown to depend critically on their subunit composition, much effort has been directed towards understanding the assembly of native receptors. This could provide a rational basis for the design of compounds able to interact with specific... 

Figure 11.9 GAB Ac receptor pharmacology and structure, (a) Various GAB Ac agonists and antagonists described in the text. Picrotoxinin is the active component of picrotoxin and also acts at GABAa receptors, (b) Presumed subunit structures of GABAc receptors. The receptors can form as homomeric assemblies of p subunits but native receptors may be heteromeric assemblies of p subunits (e.g. pi and p2) or may contain both p and y subunits...

Xu et al. [5] described the effect of (z>)-penicillamine on the binding of several antiacetylcholine receptor monoclonal antibodies to the Torpedo acetylcholine receptor. Penicillamine is covalently incorporated into the acetylcholine receptor through SS exchange at the cysteine residues of the a-subunit, altering the antigenic structure of the receptor. This effect on the structure of the native receptor at the neuromuscular junction may be responsible for the establishment of the autoimmune response to the acetylcholine receptor in (i))-penicillamine-induced myasthenia gravis. Cysteine and penicillamine interact to form penicillamine-cysteine mixed disulfide complexes [6] ... 

Simultaneously, Gorski s group (Welshons et al. 1984), utilizing a type of cell fractionation that permits separating the cytoplasm from the nucleus, was also able to detect the presence of nuclear ER, even if the cell had not been exposed to hormones. These findings led to a different theoretical formulation, according to which the native receptors would be found in the cell nucleus, to which the hormone would accede directly. 

As was previously established, the spatial structure of the receptor domains is altered by interaction with the hormone, with DNA, with other proteins, and by the state of the receptor phosphorylation. Different states of folding suppose that the receptor exhibits different surfaces that permit it to gain or to lose affinity for DNA sequences or for proteins, as they are components of the native receptor or of the transcriptional machinery. The different properties that... 

Fig. 1.5. Activation of the native receptor by thehormone. The hormone-receptor interaction determines a very strong bond that attracts distant amino acid residues, which alters the three-dimensional structure of the receptor. As a consequence, the receptor loses its affinity for the proteins that were originally close but that no longer find their zones of contact with the receptor. Simultaneously, the receptor reorganizes other hormone-dependent zones it acquires dimerization capacity and exhibits a capacity to bind to DNA and to transcription factors. The interaction with antiestrogens also produces a conformational change, which can give rise or not to the formation of dimers, in any case with a different conformation...

Dimerization is a process necessary for the receptor to carry out its interaction with DNA and to initiate the response to the hormone. Dimerization occurs when the receptor monomer has freed itself of hsp90 and the other accompanying proteins forming the structure of the native receptor. Moreover, binding to the hormone provides the receptor with the necessary three-dimensional structure to produce the interaction between the two receptor monomers (Kuiper et al. 1996 O Malley 1990). 

The Xenopus oocyte can reliably express LGIC receptors. In our laboratory, we have seen robust expression of the Torpedo nAChR, 5-HT3 receptors and various GABAa receptor subtypes in oocytes. Injection of cRNA transcripts is a convenient and reproducible way to achieve the expression levels needed for functional analysis of receptor subtypes. We have found that functional characterization with this system complements biochemical experiments conducted on native receptors or those that have been expressed in mammahan cells. A combination of these approaches is essential for furthering our understanding of structure-function relationships in these receptors. 

---