Molecular Identification and Characterization of Xenopus Egg Uroplakin III, an Egg Raft-associated Transmembrane Protein That Is Tyrosine-phosphorylated upon Fertilization

Abstract

Here we describe mass spectrometric identification, molecular cloning, and biochemical characterization of a lipid/membrane raft-associated protein that is tyrosine-phosphorylated upon Xenopus egg fertilization. This protein is homologous to mammalian uroplakin III, a member of the uroplakin family proteins (UPs) that constitute asymmetric unit membranes in the mammalian urothelial tissues, thus termed Xenopus uroplakin III (xUPIII). xUPIII contains N-linked sugars and is highly expressed in Xenopus eggs, ovary, urinary tract, and kidney. In unfertilized eggs, xUPIII is predominantly localized to the lipid/membrane rafts and exposed on the cell surface, as judged by surface biotinylation experiments and indirect immunofluorescent studies. After fertilization or hydrogen peroxide-induced egg activation, xUPIII becomes rapidly phosphorylated on tyrosine residue-249, which locates in the carboxyl-terminal cytoplasmic tail of the molecule. Raft localization and tyrosine phosphorylation of xUPIII can be reconstituted in HEK293 cells by coexpression of xUPIII, and Xenopus c-Src, a tyrosine kinase whose fertilization-induced activation in egg rafts is required for initiation of development. In mammals, UPIII is forming a complex with a tetraspanin molecule uroplakin Ib. As another tetraspanin, CD9, is known to be a critical component for sperm-egg fusion in the mouse, we have assumed that xUPIII is involved in sperm-egg interaction. An antibody against the extracellular domain of xUPIII blocks sperm-egg interaction, as judged by the occurrence of egg activation and first cell cleavage. Thus, xUPIII represents an egg raft-associated protein that is likely involved in sperm-egg interaction as well as subsequent Src-dependent intracellular events of egg activation in Xenopus.

Role of Src homology 2 domain-mediated PTK signaling in mouse zygotic development

“…This difference was not significant as determined by Mann-Whitney rank sum test (PZ0.561). These results are consistent with the recently published experiments testing these fusion proteins in eggs activated by normal fertilization (Kurokawa et al 2004a, Mehlmann & Jaffe 2005 (Sakakibara et al 2005), the sperm-induced calcium transient (Abassi et al 2000), pronuclear migration and fusion (Moore & Kinsey 1995, Wright & Schatten 1995, as well as developmental competence (Livingston et al 1998, Sharma et al 2005. The extent to which these pathways are active in mammalian fertilization is now the subject of much investigation.…”

Mutational analyses of UPIIIA, SHH, EFNB2, and HNF1β in persistent cloaca and associated kidney malformations

“…Very recently, it was reported that Xenopus UPIII protein is expressed in oocytes and acts as a receptor mediating sperm-egg interactions (35). The human Pro273Leu mutation (patient 6) is located in the cytoplasmic domain of UPIIIA (20) and it is notable that the cytoplasmic terminus of the frog homologue becomes phosphorylated during sperm-egg interaction (35). Our current study shows that persistent cloaca is only rarely associated with UPIIIA mutation.…”

“…We chose UPIIIA as a candidate since we previously found a de-novo missense UPIIIA mutation in an individual with persistent cloaca (20). Very recently, it was reported that Xenopus UPIII protein is expressed in oocytes and acts as a receptor mediating sperm-egg interactions (35). The human Pro273Leu mutation (patient 6) is located in the cytoplasmic domain of UPIIIA (20) and it is notable that the cytoplasmic terminus of the frog homologue becomes phosphorylated during sperm-egg interaction (35).…”

Phospho-Signaling at Oocyte Maturation and Fertilization: Set Up for Embryogenesis and Beyond Part II. Kinase Regulators and Substrates

“…Recently, they were identified in genital tract (Kalma et al 2009;Shapiro et al 2000) and germ cells and their function has been described in Xenopus fertilization Sakakibara et al 2005;Sato et al 2006), pathogen infection (Thumbikat et al 2009a;Thumbikat et al 2009b) and cancer (Matsumoto et al 2008). In Xenopus, UPIIIa a single transmembrane protein is tyrosine phosphorylated transiently in the cytosolic domain by a tyrosine kinase Src and this tyrosine www.intechopen.com Phospho-Signaling at Oocyte Maturation and Fertilization: Set Up for Embryogenesis and Beyond Part II.…”