Abstract
GPR22 is an orphan G protein-coupled receptor (GPCR). Since the ligand of the receptor is currently unknown, its biological function has not been investigated in depth. Many GPCRs and their intracellular effectors are targeted to cilia. Cilia are highly conserved eukaryotic microtubule-based organelles that protrude from the membrane of most mammalian cells. They are involved in a large variety of physiological processes and diseases. However, the details of the downstream pathways and mechanisms that maintain cilia length and structure are poorly understood. We show that morpholino knock down or overexpression of gpr22 led to defective left-right (LR) axis formation in the zebrafish embryo. Specifically, defective LR patterning included randomization of the left-specific lateral plate mesodermal genes (LPM) (lefty1, lefty2, southpaw and pitx2a), resulting in randomized cardiac looping. Furthermore, gpr22 inactivation in the Kupffer’s vesicle (KV) alone was still able to generate the phenotype, indicating that Gpr22 mainly regulates LR asymmetry through the KV. Analysis of the KV cilia by immunofluorescence and transmission electron microscopy (TEM), revealed that gpr22 knock down or overexpression resulted in changes of cilia length and structure. Further, we found that Gpr22 does not act upstream of the two cilia master regulators, Foxj1a and Rfx2. To conclude, our study characterized a novel player in the field of ciliogenesis.
Highlights
G-protein-coupled receptors (GPCRs) represent the largest protein superfamily in the human genome, including at least 800 receptors with a common seven-pass transmembrane domain organization
The most conserved regions were found in the transmembrane domains (TM) (88% identity), the intracellular loops (IL) (84% identity) and the C-terminal tail (86% identity)
To ensure that the MO-induced tail phenotype was caused by a specific reduction of G-Protein Coupled Receptor 22 (Gpr22) levels and not by off-target effects of the MO, we performed a rescue experiment by co-injecting fluorescein-tagged MO and capped, polyadenylated gpr22 mRNA into one cell stage zebrafish embryos
Summary
G-protein-coupled receptors (GPCRs) represent the largest protein superfamily in the human genome, including at least 800 receptors with a common seven-pass transmembrane domain organization. A large variety of ligands can bind and activate these receptors, such as light-sensitive compounds, pheromones, hormones, neurotransmitters, ions, peptides, proteins and many more. The endogenous ligands for more than 140 GPCRs remain unidentified making them ‘orphan’ receptors [1,2]. The current model of the receptor activation proposes that ligand binding induces a conformational change, changing the interaction with intracellular heterotrimeric G-proteins, consisting of a Ga and a Gbc subunit. The receptor catalyzes the exchange of GDP to GTP in the Ga subunit. GPCRs bind to b-arrestins and GPCR kinases (GRKs), which can activate G-protein independent signaling pathways, such as JNK, RhoA, MAPK or ERK [3]. GRKs and b-arrestins are involved in GPCR desensitization by preventing G-protein coupling and stimulating endocytosis of the receptor [4,5]
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