Expression and characterization of the human YWK-II gene, encoding a sperm membrane protein related to the Alzheimer betaA4-amyloid precursor protein

Abstract

The YWK-II cDNA, RSD-2, encoding a sperm membrane protein was isolated from a rat testis cDNA expression library. Using the RSD-2 insert in combination with rapid amplification of cDNA ends (RACE), the corresponding human gene was isolated from a human testis cDNA expression library. The human testis cDNA, HSD-2, is 3654 bp in length and contains an open reading frame of 763 codons. Hydropathicity analysis showed that the deduced polypeptide is a single strand transmembrane protein. The deduced polypeptide has partial homology with the amyloid precursor protein (APP) and high homology with the amyloid precursor homologue, APLP2/APPH. The YWK-II gene was mapped and assigned to human chromosome locus: 11q24-25. Northern blotting of various human tissue RNAs using the HSD-2 cDNA as a probe showed that the gene is transcribed ubiquitously. The cytoplasmic domain of HSD-2 was expressed in Escherichia coli. In-vitro studies showed that the recombinant polypeptide bound to a GTP-binding protein (G(o)) and was phosphorylated by protein kinase C and cdc2 kinase. In mammalian F11 cells, the recombinant polypeptide was found to be coupled to G(o). Thus, the YWK-II component has the characteristics of a G(o)-coupled receptor and may be involved in G(o)-mediated signal transduction pathway. Protein kinase C and cdc2 kinase may regulate this pathway in spermatozoa by phosphorylating the cytoplasmic domain of the YWK-II component.

YWK-II protein as a novel Go-coupled receptor for Müllerian inhibiting substance in cell survival

“…Apart from the previously demonstrated binding of YWK-II protein to both rMIS and G o protein (Huang et al, 2000;Tian et al, 2001), the present study has provided further evidence of the involvement of YWK-II protein in MIS-activated and G ocoupled ERK signaling pathways as well as in mediating the effect of MIS on cell survival in a CHO model and mouse sperm in vitro and in vivo.…”

“…The ovary-derived CHO cell is similar to the sperm in that it produces G o protein (Vanhauwe et al, 2002) (Huang et al, 2000), and others have shown that this G protein is connected to the ERK cascade (Kim et al, 2003). Therefore, YWK-II protein, as a receptor for MIS, may be coupled to G o protein to activate ERK.…”

“…In a previous study, we showed that the cytoplasmic segment of the YWK-II protein binds G o protein (Huang et al, 2000). When the extracellular segment of YWK-II protein was used as bait in a yeast two-hybrid system, a recombinant active MIS (303-376; rMIS) was found binding to YWK-II protein and the interaction was verified by a GST pull-down assay and by surface plasmon resonance (SPR) (Tian et al, 2001).…”

Detailed Characterization of Neuroprotection by a Rescue Factor Humanin against Various Alzheimer's Disease-Relevant Insults

“…F11 cells, the hybrid cells of a rat embryonic day 13 (E13) primary cultured neuron and a mouse neuroblastoma, were grown in Ham's F-12 (Life Technologies, Gaithersburg, MD) plus 18% fetal bovine serum (FBS) (Hyclone, L ogan, UT) and antibiotics, as described previously (Platika et al, 1985;Yamatsuji et al, 1996;Huang et al, 2000;Niikura et al, 2001). F11 cells (7 ϫ 10 4 cells per well in a six-well plate cultured in Ham's F-12 plus 18% FBS for 12-16 hr) were transfected with plasmids encoding FAD genes by lipofection [1 g of FAD cDNA, 2 l of LipofectAM I N E, and 4 l of PLUS reagent (Life Technologies)] in the absence of serum for 3 hr and were incubated with Ham's F-12 plus 18% FBS for 2 hr.…”

Molecular characterization of neurohybrid cell death induced by Alzheimer's amyloid‐β peptides via p75NTR/PLAIDD

“…F11 cells are the hybrid of a rat embryonic day 13 primary cultured neuron with a mouse neuroblastoma NTG18 and have so far been used as the best model for primary neurons (Yamatsuji et al 1996;Storms and Rutishauser 1998;Ghil et al 2000;Hagiwara et al 2000;Hashimoto et al 2000aHashimoto et al ,b, 2001aHashimoto et al ,b,c, 2003aHashimoto et al , 2004aHuang et al 2000;Sudo et al 2000Sudo et al , 2001Niikura et al 2001). F11 cells overexpressing L401K p75NTR (F11/L401K cell) were established as follows.…”