Heterodimerization Splice Variants and Isoforms

Splice variants, lacking the full complement of TMDs, have been described for many different GPCR. These truncated receptors have been reported to have a dominant negative effect on wild-type receptor function through dimerization, resulting in trapping of wild-type receptors in the ER (Benkirane et al. 1997; Karpa et al. 2000; Grosse et al. 1997). Therefore, heterodimerization between wild-type receptors and splice variants may play an important role in normal physiological regulation of receptor function and in pathological conditions.

RNA splicing results in the production of a truncated 5-HT2C receptor (5-HT2Ctr) containing the N-terminus and TMDs I-III followed by 96 unique amino acids resulting from a frame shift and premature stop codon (Canton et al. 1996). In human brain tissue, 5-HT2Ctr mRNA is found in all regions containing the full-length mRNA for the 5-HT2C receptor, including hippocampus, hypothalamus, frontal cortex, striatum, and olfactory tubercle (Canton et al. 1996). The mRNA for 5-HT2Ctr represents 60% of the total 5-HT2C mRNA found in choroid plexus and 20-30% of that found in other neuronal tissues. Western blots of membrane extracts from trans-fected NIH3T3 cells revealed immunoreactive bands the predicted size of the truncated protein, indicating that mRNA encoding 5-HT2Ctr is translated into protein. Radioligand binding and inositol phosphate production were not observed in the transfected cells, indicating that 5-HT2Ctr is nonfunctional (Canton et al. 1996).

Fluorescence confocal microscopy was used to compare the trafficking patterns of YFP-tagged full-length and splice variant 5-HT2C receptors. The full-length receptor was expressed on the plasma membrane, while the splice variant was retained inside the cell and did not traffic to the plasma membrane (Fig. 7.8). Cotransfection of HEK 293 cells with 5-HT2Ctr/CFP and ER-YFP or Golgi-YFP markers revealed that 5-HT2Ctr was retained in the ER and did not traffic to the Golgi. When coexpressed with 5-HT2Ctr the full-length 5-HT2C receptors were retained inside the cell and a decrease in cell surface 5-HT2C receptor expression level was observed in an intact cell radioligand binding assay (Fig. 7.9). In cells coexpressing 5-HT2C/CFP and 5-HT2Ctr/YFP the mean FRET efficiency was 24.6 ± 1.3% (n = 35). Negligible FRET was observed in 10 cells expressing 5-HT2Ctr/ CFP alone (-2.7 ± 1.2%) or in combination with the ER-YFP marker (4.2 ± 2.3%). These results are consistent with the hypothesis that 5-HT2Ctr can associate with and inhibit plasma membrane targeting of 5-HT2C receptors.

Alternative splicing of 5-HT2A and 5-HT6 RNA results in the synthesis of proteins that are truncated following TMD III (5-HT2Atr and 5-HT6tr), and these truncated receptors are nonfunctional (Olsen et al. 1999; Guest et al. 2000). In contrast to our studies with 5-HT2Ctr, splice variants of 5-HT2A and 5-HT6 receptors have been reported to be expressed on cell membranes and not to interfere with wildtype receptor function (Olsen et al. 1999; Guest et al. 2000). However, these conclusions were based on the results of Western blots, and confocal microscopy was not performed to identify the trafficking pattern of these splice variants or to confirm plasma membrane expression. While coexpression studies indicate that 5-HT2Ctr forms heterodimers with full-length 5-HT2C receptors within the ER, thereby inhibiting normal trafficking of 5-HT2C receptors to the plasma membrane, the functional significance of this observation remains unknown. Additional studies demonstrating a change in 5-HT2C receptor expression or function in primary cultures coexpressing native splice variant and full-length receptors will be required to address this issue. At present, there are no specific antibodies for 5-HT2Ctr to verify expression levels in primary cultures or to demonstrate coimmunoprecipitation of endogenously expressed native and truncated 5-HT2C receptors.

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Defeat Drugs and Live Free

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