Dominant Negative Effects

Loss of receptor function often becomes apparent only when both alleles are affected. In some cases, however, impaired trafficking of the wild-type receptor to the plasma membrane or defects in signal transduction is caused by dominant-negative effects of coexpressed mutated receptors in the heterozygous state (for review, see Reference 4). This has recently been postulated to explain the dominant mode of inheritance in adRP [234]. Expression of the Class II rhodopsin mutants P23H and G188R was shown to interfere with the biosyn-thetic maturation of coexpressed wild-type rhodopsin, leading to retention in the ER. In contrast, ER retention of wild-type rhodopsin was not seen when coexpressed with the Class I mutant, V345M rhodopsin. Direct, physical interaction between mutant and wild-type rhodopsins was responsible for ER retention and premature proteasome degradation of the wild-type protein [234] . These observations confirmed and extended earlier observations seen in Drosophila and mammalian expression systems [124, 235, 236], and further support the potential for rhodopsin dimerization during biosynthesis, trafficking, and signal transduction [237, 238], similar to the role played by dimeriza-tion with other GPCRs (for review, see Reference 239). While compelling, the dominant-negative effect is not necessarily mutually exclusive with other mechanisms that may also be at play in RP, such as the toxic gain of function shown for some mutant forms of rhodopsin that lead to photoreceptor death as described above. Similar to rhodopsin, GnRHR has been shown to dimerize as part of the process of receptor activation [240] . Cotransfection of mutant and wild-type GnRHRs in vitro revealed that the mutated receptors exert a dominant-negative effect on wild-type receptor with respect to ligand binding and intracellular signaling [163, 241, 242] as a result of w^^pe receptor retention in the ER by association with trafficking - defective mutant receptors [241]. This phenomenon has also been observed for other GPCRs, including a naturally occurring mutant form of MC4R that has been shown to exert a dominant-negative effect on wild-type receptor, potentially through dimeriza-tion [243] . It will be necessary to better understand the effects of pharmacological chaperones on dominant-negative effects, such that facilitation of mutant receptor export from the ER also permits trafficking of wild 4ype receptors to the cell surface.

Similar to the dominant-negative effects on wild-type receptors, evidence now suggests that coexpression of two mutant forms of a GPCR are functionally unique and may respond differently to a pharmacological chaperone than either mutant form expressed alone. This has been clearly shown by the different effects of pharmacological chaperones on coexpressed pairs of GnRHR mutants n vitro as discussed above [165, 166] . In these cases, the [n vitro response of the combination of mutant receptors to a pharmacological chap-erone would be expected to be more predictive of the n vivo response to a pharmacological chaperone than the in vitro response of each individual mutant receptor alone. Thus, a pharmacogenetic approach that involves screening for responsive mutations in a well-designed in vitro assay, whether alone or in combination with wild-type or other mutant receptor forms, is likely to help select patients that harbor mutations or combinations of mutations that may be responsive [n vivo. Ideally, for homozygous autosomal recessive or autosomal dominant diseases caused by mutated GPCRs, an in vitro response will likely enrich for the population of patients harboring mutations that may be responsive in vivo. Similarly, for autosomal recessive diseases in which patients are most likely to be heterozygous for two different GPCR mutations, the pharmacogenetic approach would be most useful if the in vitro response of the combination of mutant receptors was assessed. Finally, the correlation of the in vitro and in vivo responses to a pharmacological chaperone may apply only to the biochemical measures of receptor cell surface expression, ligand binding, and intracellular signaling. Predictive value for clinical improvement could be achieved only if the in vitro response was positively correlated with improved clinical symptoms or decreased disease severity.

0 0

Post a comment