Abstract
Lysophosphatidic acid (LPA) induces diverse biological responses in many types of cells and tissues by activating its specific G protein-coupled receptors (GPCRs). Previously, three cognate LPA GPCRs (LP(A1)/VZG-1/EDG-2, LP(A2)/EDG-4, and LP(A3)/EDG-7) were identified in mammals. By contrast, an unrelated GPCR, PSP24, was reported to be a high affinity LPA receptor in Xenopus laevis oocytes, raising the possibility that Xenopus uses a very different form of LPA signaling. Toward addressing this issue, we report two novel Xenopus genes, xlp(A1)-1 and xlp(A1)-2, encoding LP(A1) homologs (approximately 90% amino acid sequence identity with mammalian LP(A1)). Both xlp(A1)-1 and xlp(A1)-2 are expressed in oocytes and the nervous system. Overexpression of either gene in oocytes potentiated LPA-induced oscillatory chloride ion currents through a pertussis toxin-insensitive pathway. Injection of antisense oligonucleotides designed to inhibit xlp(A1)-1 and xlp(A1)-2 expression in oocytes eliminated their endogenous response to LPA. Furthermore, retrovirus-mediated heterologous expression of xlp(A1)-1 or xlp(A1)-2 in B103 rat neuroblastoma cells that are unresponsive to LPA conferred LPA-induced cell rounding and adenylyl cyclase inhibition. These results indicate that XLP(A1)-1 and XLP(A1)-2 are functional Xenopus LPA receptors and demonstrate the evolutionary conservation of LPA signaling over a range of vertebrate phylogeny.
Highlights
The nucleotide sequences reported in this paper have been submitted to the EMBL/GenBankTM/EBI Data Bank with accession numbers AJ249843 and AJ249844
Isolation of xlpA1-1 and xlpA1-2—To identify novel G protein-coupled receptors (GPCRs) in Xenopus oocytes, we performed a polymerase chain reaction (PCR)-based screen using degenerate oligonucleotide primers designed against transmembrane domains (TMD) II and VII [6, 21]
DNA sequencing identified two fragments with 90% identity in predicted amino acid sequences to those of the mammalian LPA1 receptor. These PCR fragments were used as probes to screen a Xenopus oocyte cDNA library, and two different cDNAs were cloned, which encoded GPCRs consisting of 366 amino acids that differed by 6 amino acids (Fig. 1, A and B)
Summary
Materials—[␣-32P]deoxy CTP was purchased from PerkinElmer Life Sciences. LPA (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate) was purchased from Avanti Polar Lipids (Alabaster, AL). CRNA injection, and electrophysiology were performed as described previously [22]. Retrovirus Systems—The entire ORFs for xlpA1-1 and -2 were subcloned into HindIII and XbaI sites of a pFLAG-CMV-1 mammalian expression vector (Eastman Kodak Co.) to introduce preprotrypsinleader/FLAG-tag sequences into amino-terminal extracellular regions of each receptor for immunocytochemical detection of the receptor proteins. These constructs were subcloned into BamHI and XhoI sites of a Moloney murine leukemia retroviral vector, LZRS-EGFP [24].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
