Splice variants of G protein-coupled receptors

G protein-coupled receptors (GPCRs) are encoded by a vast gene superfamily, reflecting the large number of ligands that must be specifically recognized at any given cell surface. The discovery that the variety of GPCRs is further expanded through the generation of splice variants was therefore somewhat surprising. Studies of the functional consequences of alternative splicing have focused on ligand binding, signaling, constitutive activity, and downregulation. However, GPCRs also appear to interact directly with many other intracellular proteins in addition to G proteins. Intriguingly, the domains involved in these interactions are the predominant sites of variation arising through splicing.     

A novel splice variant of Gαq-coupled Bombyx CAPA-PVK receptor 1 functions as a specific Gαi/o-linked receptor for CAPA-PK

Alternative splicing enables G protein-coupled receptor (GPCR) genes to greatly increase the number of structurally and functionally distinct receptor isoforms. However, the functional role and relevance of the individual GPCR splice variants in regulating physiological processes are still to be assessed. A naturally occurring alternative splice variant of Bombyx CAPA-PVK receptor, BomCAPA-PVK-R1-Δ341, has been shown to act as a dominant-negative protein to regulate cell surface expression and function of the canonical CAPA-PVK receptor. Herein, using functional assays, we identify the splice variant Δ341 as a specific receptor for neuropeptide CAPA-PK, and upon activation, Δ341 signals to ERK1/2 pathway. Further characterization demonstrates that Δ341 couples to Gαi/o, distinct from the Gαq-coupled canonical CAPA-PVK receptor, triggering ERK1/2 phosphorylation through Gβγ-PI3K-PKCζ signaling cascade. Moreover, our ELISA data show that the ligand-dependent internalization of the splice variant Δ341 is significantly impaired due to lack of GRKs-mediated phosphorylation sites. Our findings highlight the potential of this knowledge for molecular, pharmacological and physiological studies on GPCR splice variants in the future.     

Differential trafficking of GluR7 kainate receptor subunit splice variants

Kainate receptors (KARs) are heteromeric ionotropic glutamate receptors that play a variety of roles in the regulation of synaptic network activity. The function of glutamate receptors (GluRs) is highly dependent on their surface density in specific neuronal domains. Alternative splicing is known to regulate surface expression of GluR5 and GluR6 subunits. The KAR subunit GluR7 exists under different splice variant isoforms in the C-terminal domain (GluR7a and GluR7b). Here we have studied the trafficking of GluR7 splice variants in cultured hippocampal neurons from wild-type and KAR mutant mice. We have found that alternative splicing regulates surface expression of GluR7-containing KARs. GluR7a and GluR7b differentially traffic from the ER to the plasma membrane. GluR7a is highly expressed at the plasma membrane, and its trafficking is dependent on a stretch of positively charged amino acids also found in GluR6a. In contrast, GluR7b is detected at the plasma membrane at a low level and retained mostly in the endoplasmic reticulum (ER). The RXR motif of GluR7b does not act as an ER retention motif, at variance with other receptors and ion channels, but might be involved during the assembly process. Like GluR6a, GluR7a promotes surface expression of ER-retained subunit splice variants when assembled in heteromeric KARs. However, our results also suggest that this positive regulation of KAR trafficking is limited by the ability of different combinations of subunits to form heteromeric receptor assemblies. These data further define the complex rules that govern membrane delivery and subcellular distribution of KARs.     

PNRC1剪接变异体的鉴定及其在辅激活核受体介导基因转录功能上的比较

为分析富含脯氨酸核受体辅调节蛋白1(PNRC1)选择性剪接, 及比较PNRC1剪接变异体在辅激活核受体介导基因转录功能上的差异,在生物信息学方法分析PNRC1剪接变异体的基础上,设计一定的特异性引物,采用RT-PCR结合克隆测序的方法对这些剪接变异体进行验证. 利用酵母双杂交和荧光素酶报告系统实验,分析它们与核受体的相互作用及比较它们在辅激活核受体介导基因转录功能上的差异.结果显示,生物信息学预测的几个剪接变异体真实存在于人的组织和细胞系中,这些剪接变异体在与雌激素受体α(ERα)、类固醇衍生因子1(SF1)等核受体的相互作用的强度及辅激活核受体介导基因转录功能上存在较大的差异. 研究提示,PNRC1这些剪接变异体在体内可能发挥不同的功能.     

Expression of splice variants of 1alpha-hydroxylase in mcf-7 breast cancer cells

1,25-Dihydroxyvitamin D(3) (calcitriol) is the most active natural metabolite of Vitamin D(3). It has strong antiproliferative and differentiating effects on various cell types including breast cancer cells. 25-Hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase, CYP27B1) is one of the key enzymes in the formation of calcitriol. It has been found in breast cancer cells suggesting an autocrine regulation of formation of calcitriol in these cells. Alternative splicing of the encoding genes for this enzyme can possibly play a role in regulating the enzyme level and can explain tissue specific variations of 1alpha-hydroxylase activity. Splice variants containing intron 1 may encode for truncated proteins with deletion of protein domains which are essential for its enzymatic activity. In order to obtain more information on the abundance of 1alpha-hydroxylase splice variants, we performed a highly specific nested touchdown PCR in MCF-7 cells. The full-length sequence of 1alpha-hydroxylase and two different splice variants of this enzyme containing intron 1 were isolated. By Western blot technique we then confirmed the protein products of the full-length enzyme and its splice variants. We hypothesize that that the expression of splice variants can lead to a quantitatively lower expression of the mRNA of the full-length enzyme. The abundance of less active 1alpha-hydroxylase protein variants can alter the local synthesis of calcitriol in the cells and may explain variations of enzymatic activity in different cells and tissues.     

Synthesis and Characterization of Azido Aryl Analogs of IBNtxA for Radio-Photoaffinity Labeling Opioid Receptors in Cell Lines and in Mouse Brain

Mu opioid receptors (MOR-1) mediate the biological actions of clinically used opioids such as morphine, oxycodone, and fentanyl. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating multiple splice variants. One type of splice variants are truncated variants containing only six transmembrane domains (6TM) that mediate the analgesic action of novel opioid drugs such as 3′-iodobenzoylnaltrexamide (IBNtxA). Previously, we have shown that IBNtxA is a potent analgesic effective in a spectrum of pain models but lacks many side-effects associated with traditional opiates. In order to investigate the targets labeled by IBNtxA, we synthesized two arylazido analogs of IBNtxA that allow photolabeling of mouse mu opioid receptors (mMOR-1) in transfected cell lines and mMOR-1 protein complexes that may comprise the 6TM sites in mouse brain. We demonstrate that both allyl and alkyne arylazido derivatives of IBNtxA efficiently radio-photolabeled mMOR-1 in cell lines and MOR-1 protein complexes expressed either exogenously or endogenously, as well as found in mouse brain. In future, design and application of such radio-photolabeling ligands with a conjugated handle will provide useful tools for further isolating or purifying MOR-1 to investigate site specific ligand–protein contacts and its signaling complexes.

     

Stabilization of the μ-Opioid Receptor by Truncated Single Transmembrane Splice Variants through a Chaperone-like Action

The μ-opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, as illustrated by the identification of an array of splice variants generated by both 5′ and 3′ alternative splicing. The current study reports the identification of another set of splice variants conserved across species that are generated through exon skipping or insertion that encodes proteins containing only a single transmembrane (TM) domain. Using a Tet-Off system, we demonstrated that the truncated single TM variants can dimerize with the full-length 7-TM μ-opioid receptor (MOR-1) in the endoplasmic reticulum, leading to increased expression of MOR-1 at the protein level by a chaperone-like function that minimizes endoplasmic reticulum-associated degradation. In vivo antisense studies suggested that the single TM variants play an important role in morphine analgesia, presumably through modulation of receptor expression levels. Our studies suggest the functional roles of truncated receptors in other G protein-coupled receptor families.     

Functional significance of cleavable signal peptides of G protein-coupled receptors

About 5-10% of the G protein-coupled receptors (GPCRs) contain N-terminal signal peptides that are cleaved off by the signal peptidases of the endoplasmic reticulum (ER) during the translocon-mediated receptor insertion into the ER membrane. The reason as to why only a subset of the GPCRs requires these additional signal peptides was addressed in the past decade only by a limited number of studies. Recent progress suggests that signal peptides of GPCRs do not only serve the classical ER targeting and translocon gating functions as described for the signal peptides of secretory proteins. In the case of GPCRs, uncleaved pseudo signal peptides may regulate receptor expression at the plasma membrane and may also influence G protein coupling. Moreover, signal peptides of GPCRs seem to match functionally with sequences of the mature N tails. In this review, we summarize the current knowledge about cleavable signal peptides of GPCRs and address the question whether these sequences may be future drug targets in pharmacology.