CT Gene PRTD-NY2 Function

Processes of germ-cell development and tumor development shareimportant similarities. One of the most significant marks is the discoveryof a growing number of the cancer/testis genes. Finding and investigatingCT genes will further elucidate the mechanism of spermatogenesis andtumorigenesis.PRTD-NY2 is a new gene cloned by our lab from human adult testis.The bioimformatics analysis showed that it is a RGS protein belongs toF/RL family. And its chromosome location is associated with many kindsof tumors. In this research, we try to figure out the role of PRTD-NY2 inspermatogenesis and tumorigensis, and its mechanism.First of all, we found PRTD-NY2 is a CT gene by RT-PCR. Thehuman testis immunohistochemistry analysis showed that PRTD-NY2 waslocated in the cytoplasm of spermatogonia, spermatocytes and spermatids,and also in the nucleus of spermatids. PRTD-NY2 was found in thepost-nuclear region and the residual body of the spermatozoa. The resultsof PRTD-NY2 mRNA expression in azoospermatism indicated that it mayproduce a marked effect in the post-meiosis stages.RGS proteins directly bound to activated Gαsubunits to transferthem into inactivate forms and down regulate the GPCR mediatedsignaling. So, we verified that PRTD-NY2 could interact with Gα12/13and Gαq in human testis. As a RGS protein, PRTD-NY2 plays roles in spermatogenesis by regulating these G-protein-αsubunits.In the study of Gα12 in spermatogenesis, we found Gα12 wasexpressed in the cytoplasm of Leydig cells and spermatid from theelongating Sb phase to mature sperm. Indirect immunofluorescence ofhuman sperm revealed the presence of Gα12 in the neck region and themidpiece of the sperm. Gα12 in spermatids and spermatozoa partialco-localized with F-actin andα-tubulin. Gα12 plays a role in thecytoskeleton reorganization for sperm polarity and tail formation mediatedby F-actin andα-tubulin. The expression of Gα12 in the testis ofnon-obstructive azoospermatism showed abnormal Gα12 expression mayeone of the causes of spermatogenesis handicap. The defective expressionof Gα12 in low motility spermatozoa with midpieces that were bent onthemselves suggests that the cause of low motility may be a consequenceof the abnormal quantity or location of Gα12 produced in the first twophase of spermiogenesis and/or abnormal reservation in the last one.PRTD-NY2 is expressed in the cytoplasm of spermatids, and it caninteract with Gα12, so it probably involved in the cytoskeletonreorganization for sperm polarity and tail formation.Subsequently, we also investigated the role of Gα13 in speratogenesis.We found there is Gα13 expression in human testis and sperm. And, Gα13expressed in mouse testis and sperm with the same molecular mass assomatic tissues. It is expressed in the cytoplasm of human and mousespermatogonia and spermatocytes, but in the nucleus of elongating spermatids. We also observed the transformation of Gα13 from thecytoplasm of round spermatids to the nucleus of elongating and elongatedspermatids by mouse spermatogenic cells immunofluorescene analysis.Indirect immunofluorescence of human spermatozoa confirmed thepresence of Gα13 in the nucleus. All these show the location of Gα13 inspermatids is different in the somatic cells. Gα13's expression in largeheads is defective. Gα13 is associated with sperm nuclear shaping. Wealso found the localization of PRTD-NY2 is similar to Gα13. Furthermore,the subcellular position of PRTD-NY2 fragments indicated the nuclearlocalization signaling of PRTD-NY2 was at its N-terminal. So, testisspecific PRTD-NY2 may co-migrate with Gα13 from cytoplasm tonucleus during spermiogensis and involve in nuclear compaction.At last, we investigated the role of PRTD-NY2 in tumorigenesis andit mechanism. The analysis of human tumor tissue microarray with 96patients showed that PRTD-NY2 was expressed in carcinomas (major insquamous carcinoma and adenocacinoma). The immunohistochemistry oftumor tissues of 14 squamous carcinoma and adenocacinoma patients andesophageal carcinoma tissue microarray, RT-PCR and Western Blot of 4kinds of cancer cell lines with different migratory ability, and themetastasis of PRTD-NY2 over expressed and RNAi cancer cell lines innude mice, all indicated PRTD-NY2 could suppress metastasis.PRTD-NY2 could be translated into proteins with differate molecularmasses by translating from different start sites in E. coli and many kinds of tumor cell lines. PRTD-NY2 could be translated into 146KD and 76KDproteins. They could inhibit the nude mice intraabdominal implantationmetastasis of tumor cells and distant metastasis to lung. PRTD-NY2 hasno effect on tumor cell adhension. The wound healing and transwell testsboth proved that PRTD-NY2 could depress tumor cell migrationstimulated by LPA. Pull down of tumor cells and LPA stimulated cellshaping confirmed that PRTD-NY2 could inhibit Gα12/13 mediated LPAstimulated stress fiber formation. The wound healing and transwell testsalso found 76KD fragment of PRTD-NY2 only still could depress tumorcell migration stimulated by LPA. So, 76KD fragment may be principal tometastasis.In brief, PRTD-NY2 is a new CT gene. It can interacte with Gα12/13and Gαq in human testis. It involves in sperm polarity and tail formationby regulating Gα12. Moreover, it may co-migrate with Gα13 fromcytoplasm to nucleus during spermiogensis and involve in nuclearcompaction. In prokaryon and eukaryon, PRTD-NY2 could be translatedinto proteins with differate molecular masses by translating from differentstart sites. PRTD-NY2 could inhibit Gα12/13 mediated LPA stimulatedstress fiber formation and suppress the tumor cell migration andmetastasis.