Recent molecular characterization of cloned protein genes draws attention to alternative splicing as a source of structural and functional diversity. The amino acid sequences of most GPCRs are encoded by intronless single-copy genes (regarding the coding region). However, a number of GPCR genes show an exon/intron assembly of their coding regions, as described for rhodopsin, some amine and peptide receptors, and GPCRs with a large extracellular domain (glycoprotein hormone receptors, many Family 2 GPCRs). The existence of introns in GPCR genes provides the potential for additional diversity by virtue of alternative splicing events which may generate distinct receptor isoforms. For example, pharmacological and molecular biological studies have resulted in the cloning of cDNAs encoding four EP prostanoid receptors. The cloning of these receptors has revealed further heterogeneity due to alternative mRNA splicing. Specifically, eight human EP3 receptor isoforms have been identified which differ only in their C termini (Pierce and Regan 1998). It should be noted that a tissue-specific occurrence of distinct splice variants has been described, for example, for the PACAP receptor (Chatterjee etal. 1996) and the corticotropin releasing factor (CRF) receptor (Ardati et al. 1999).
The genomic intron/exon structure and even the number of subtypes of a given GPCR are not necessarily conserved among species. Extensive studies on opsin genes have shown that introns in the coding region can appear and disappear during evolution. For example, human rhodopsin is encoded by four exons, but in some fish species the coding region for rhodopsin is intronless (Venkatesh et al. 1999). At least in principle, this implicates species-specific differences in the isoform pattern generated by alternative splicing events.
Two functional types of GPCR splice variants can be distinguished. First, usage of an alternative splice site can generate a functional receptor as demonstrated for a large number of GPCRs such as the mGluR1 (Prezeau et al. 1996) and the D2 dopamine receptor (Seeman et al. 2000). In most cases, the divergence between receptor isoforms is limited to the C-terminal tail, a region involved in internalization, down-regulation and interactions with other proteins. As shown for the prostanoid EP3 and 5-HT4 receptor isoforms, alternative splice products can vary in their basal activity when expressed in vitro. The extent of constitutive activity was found to be reversally correlated with the length of the C-terminal portion of the splice variants (Jin et al. 1997; Claeysen et al. 1999). Second, an improper splicing event can produce a non-functional receptor protein that may display dominant negative effects on the wild-type receptor. It has been demonstrated that the expression of a truncated isoform of the gonadotropin-releasing hormone (GnRH) receptor can decrease the signalling efficacy of the full-length receptor by reducing its cell surface expression levels (Grosse et al. 1997). This dominant negative effect was highly specific for the GnRH receptor and was probably due to heterocomplex formation between the two proteins. One may speculate that co-expression of truncated receptor isoforms may modulate the gon-adotropes' responsiveness to GnRH and thus contribute to the fine tuning of gonadotropin release in vivo. Similarly, the ability of an EP1 receptor isoform to inhibit signalling by EP1 as well as EP4 receptors can be explained by complex formation between these different receptors (Okuda-Ashitaka et al. 1996). Impaired insertion of the wild-type receptor into the plasma membrane is a common mechanism underlying the dominant negative effects of co-expressed mutant or truncated receptors. It was demonstrated that a naturally occurring allele coding for a truncated CCR5 chemokine receptor which functions as a co-receptor for infection by primary M-tropic HIV-1 strains exerts a dominant negative effect on the viral env protein-mediated cell fusion (Samson et al. 1996). It was later shown that the truncated receptor complexes with the wild-type CCR5 and that this interaction retains CCR5 in the endoplasmic recticulum (ER) resulting in reduced cell surface expression (Benkirane et al. 1997). In addition, defective intracellular transport due to the formation of misfolded complexes between wild-type and mutant rhodopsin in the ER is held responsible for the dominant effect of one mutant allele in case of retinal degeneration in Drosophila (Colley et al. 1995). It should be noted that dominant negative effects of mutant GPCRs on wild-type receptor function can also be caused by other mechanisms such as titrating G proteins away from the wild-type receptor (Leavitt et al. 1999). Thus, expression of truncated or modified receptor proteins may highlight a novel principle of specific modulation of GPCR function.
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