cAMP-Epac2-mediated activation of rapl in developing male germ cells: RA-RhoGAP as a possible direct down-stream effector

Evanthia Aivatiadou, Michela Ripolone, Francesco Brunetti, Giovanna Berruti

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Rap1 is a small GTPase that functions as a positional signal and organizer of cell architecture. Recently Rap1 is emerged to play a critical role during sperm differentiation since its inactivation in haploid cells leads to a premature release of spermatids from the supporting Sertoli cell resulting in male infertility. How Rap1 is activated in spermatogenic cells has not yet been determined. Our objective was to investigate on a possible cAMP-mediated activation of Rap1 employing a cAMP analogue selective to Epac, the Rap1 activator directly responsive to cAMP, for stimulating cultured testis germ cells. Here we provide biochemical, cellular and functional evidence that the Epac variant known as Epac2 is expressed as both a transcript and a protein and that it is able to promote Rap1 activation in the cultured cells. A time course immunofluorescence analysis carried out on stimulated cells revealed the translocation of endogenous Epac2, which is cytosolic, towards the site where Rap1 is located, i.e., the Golgi complex, thus documenting the effective Rap1-Epac2 protein interaction 'in vivo' leading to Rap1-GTP loading. A combination of biochemical and molecular techniques supported the immunofluorescence data. The search for the presence of a putative Rap1 downstream effector, described in differentiating somatic cells as a target of cAMP-Epac-activated Rap1, revealed the presence in spermatogenic cells of RA-RhoGAP, a Rap1-activated Rho GTPase- activating protein. Taken together, our results, obtained with endogenously expressed proteins, are consistent with a cAMP/Epac2/Rap1-mediated signaling that could exert its action, among others, through RA-RhoGAP to promote the progression of spermatogenesis.

Original languageEnglish
Pages (from-to)407-416
Number of pages10
JournalMolecular Reproduction and Development
Volume76
Issue number4
DOIs
Publication statusPublished - Apr 2009

Fingerprint

Germ Cells
Fluorescent Antibody Technique
Proteins
Spermatids
Monomeric GTP-Binding Proteins
Sertoli Cells
Haploidy
Male Infertility
Golgi Apparatus
Spermatogenesis
Guanosine Triphosphate
Spermatozoa
Testis
Cultured Cells

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology
  • Cell Biology

Cite this

cAMP-Epac2-mediated activation of rapl in developing male germ cells : RA-RhoGAP as a possible direct down-stream effector. / Aivatiadou, Evanthia; Ripolone, Michela; Brunetti, Francesco; Berruti, Giovanna.

In: Molecular Reproduction and Development, Vol. 76, No. 4, 04.2009, p. 407-416.

Research output: Contribution to journalArticle

@article{d89f965fbd584a3591c87d48cdb8cffb,
title = "cAMP-Epac2-mediated activation of rapl in developing male germ cells: RA-RhoGAP as a possible direct down-stream effector",
abstract = "Rap1 is a small GTPase that functions as a positional signal and organizer of cell architecture. Recently Rap1 is emerged to play a critical role during sperm differentiation since its inactivation in haploid cells leads to a premature release of spermatids from the supporting Sertoli cell resulting in male infertility. How Rap1 is activated in spermatogenic cells has not yet been determined. Our objective was to investigate on a possible cAMP-mediated activation of Rap1 employing a cAMP analogue selective to Epac, the Rap1 activator directly responsive to cAMP, for stimulating cultured testis germ cells. Here we provide biochemical, cellular and functional evidence that the Epac variant known as Epac2 is expressed as both a transcript and a protein and that it is able to promote Rap1 activation in the cultured cells. A time course immunofluorescence analysis carried out on stimulated cells revealed the translocation of endogenous Epac2, which is cytosolic, towards the site where Rap1 is located, i.e., the Golgi complex, thus documenting the effective Rap1-Epac2 protein interaction 'in vivo' leading to Rap1-GTP loading. A combination of biochemical and molecular techniques supported the immunofluorescence data. The search for the presence of a putative Rap1 downstream effector, described in differentiating somatic cells as a target of cAMP-Epac-activated Rap1, revealed the presence in spermatogenic cells of RA-RhoGAP, a Rap1-activated Rho GTPase- activating protein. Taken together, our results, obtained with endogenously expressed proteins, are consistent with a cAMP/Epac2/Rap1-mediated signaling that could exert its action, among others, through RA-RhoGAP to promote the progression of spermatogenesis.",
author = "Evanthia Aivatiadou and Michela Ripolone and Francesco Brunetti and Giovanna Berruti",
year = "2009",
month = "4",
doi = "10.1002/mrd.20963",
language = "English",
volume = "76",
pages = "407--416",
journal = "Molecular Reproduction and Development",
issn = "1040-452X",
publisher = "Wiley-Liss Inc.",
number = "4",

}

TY - JOUR

T1 - cAMP-Epac2-mediated activation of rapl in developing male germ cells

T2 - RA-RhoGAP as a possible direct down-stream effector

AU - Aivatiadou, Evanthia

AU - Ripolone, Michela

AU - Brunetti, Francesco

AU - Berruti, Giovanna

PY - 2009/4

Y1 - 2009/4

N2 - Rap1 is a small GTPase that functions as a positional signal and organizer of cell architecture. Recently Rap1 is emerged to play a critical role during sperm differentiation since its inactivation in haploid cells leads to a premature release of spermatids from the supporting Sertoli cell resulting in male infertility. How Rap1 is activated in spermatogenic cells has not yet been determined. Our objective was to investigate on a possible cAMP-mediated activation of Rap1 employing a cAMP analogue selective to Epac, the Rap1 activator directly responsive to cAMP, for stimulating cultured testis germ cells. Here we provide biochemical, cellular and functional evidence that the Epac variant known as Epac2 is expressed as both a transcript and a protein and that it is able to promote Rap1 activation in the cultured cells. A time course immunofluorescence analysis carried out on stimulated cells revealed the translocation of endogenous Epac2, which is cytosolic, towards the site where Rap1 is located, i.e., the Golgi complex, thus documenting the effective Rap1-Epac2 protein interaction 'in vivo' leading to Rap1-GTP loading. A combination of biochemical and molecular techniques supported the immunofluorescence data. The search for the presence of a putative Rap1 downstream effector, described in differentiating somatic cells as a target of cAMP-Epac-activated Rap1, revealed the presence in spermatogenic cells of RA-RhoGAP, a Rap1-activated Rho GTPase- activating protein. Taken together, our results, obtained with endogenously expressed proteins, are consistent with a cAMP/Epac2/Rap1-mediated signaling that could exert its action, among others, through RA-RhoGAP to promote the progression of spermatogenesis.

AB - Rap1 is a small GTPase that functions as a positional signal and organizer of cell architecture. Recently Rap1 is emerged to play a critical role during sperm differentiation since its inactivation in haploid cells leads to a premature release of spermatids from the supporting Sertoli cell resulting in male infertility. How Rap1 is activated in spermatogenic cells has not yet been determined. Our objective was to investigate on a possible cAMP-mediated activation of Rap1 employing a cAMP analogue selective to Epac, the Rap1 activator directly responsive to cAMP, for stimulating cultured testis germ cells. Here we provide biochemical, cellular and functional evidence that the Epac variant known as Epac2 is expressed as both a transcript and a protein and that it is able to promote Rap1 activation in the cultured cells. A time course immunofluorescence analysis carried out on stimulated cells revealed the translocation of endogenous Epac2, which is cytosolic, towards the site where Rap1 is located, i.e., the Golgi complex, thus documenting the effective Rap1-Epac2 protein interaction 'in vivo' leading to Rap1-GTP loading. A combination of biochemical and molecular techniques supported the immunofluorescence data. The search for the presence of a putative Rap1 downstream effector, described in differentiating somatic cells as a target of cAMP-Epac-activated Rap1, revealed the presence in spermatogenic cells of RA-RhoGAP, a Rap1-activated Rho GTPase- activating protein. Taken together, our results, obtained with endogenously expressed proteins, are consistent with a cAMP/Epac2/Rap1-mediated signaling that could exert its action, among others, through RA-RhoGAP to promote the progression of spermatogenesis.

UR - http://www.scopus.com/inward/record.url?scp=65449172899&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=65449172899&partnerID=8YFLogxK

U2 - 10.1002/mrd.20963

DO - 10.1002/mrd.20963

M3 - Article

C2 - 18937323

AN - SCOPUS:65449172899

VL - 76

SP - 407

EP - 416

JO - Molecular Reproduction and Development

JF - Molecular Reproduction and Development

SN - 1040-452X

IS - 4

ER -