CABYR isoforms expressed in late steps of spermiogenesis bind with AKAPs and ropporin in mouse sperm fibrous sheath.
BACKGROUND
CABYR is a polymorphic calcium-binding protein of the sperm fibrous sheath (FS) which gene contains two coding regions (CR-A and CR-B) and is tyrosine as well as serine/threonine phosphorylated during in vitro sperm capacitation. Thus far, the detailed information on CABYR protein expression in mouse spermatogenesis is lacking. Moreover, because of the complexity of this polymorphic protein, there are no data on how CABYR isoforms associate and assemble into the FS.


METHODS
The capacity of mouse CABYR isoforms to associate into dimers and oligomers, and the relationships between CABYR and other FS proteins were studied by gel electrophoresis, Western blotting, immunofluorescence, immunoprecipitation and yeast two-hybrid analyses.


RESULTS
The predominant form of mouse CABYR in the FS is an 80 kDa variant that contains only CABYR-A encoded by coding region A. CABYR isoforms form dimers by combining the 80 kDa CABYR-A-only variant with the 50 kDa variant that contains both CABYR-A and CABYR-B encoded by full length or truncated coding region A and B. It is proposed that this step is followed by the formation of larger oligomers, which then participate in the formation of the supramolecular structure of the FS in mouse sperm. The initial expression of CABYR occurs in the cytoplasm of spermatids at step 11 of spermiogenesis and increases progressively during steps 12-15. CABYR protein gradually migrates into the sperm flagellum and localizes to the FS of the principal piece during steps 15-16. Deletion of the CABYR RII domain abolished the interaction between CABYR and AKAP3/AKAP4 but did not abolish the interaction between CABYR and ropporin suggesting that CABYR binds to AKAP3/AKAP4 by its RII domain but binds to ropporin through another as yet undefined region.


CONCLUSIONS
CABYR expresses at the late stage of spermiogenesis and its isoforms oligomerize and bind with AKAPs and ropporin. These interactions strongly suggest that CABYR participates in the assembly of complexes in the FS, which may be related to calcium signaling.
Li
Yan-Feng
YF
Department of Urology, Daping Hospital, Institute of Surgery Research, Third Military Medical University, Chongqing, PR China. lyf1000@yahoo.com.cn
He
Wei
W
Kim
Young-Hwan
YH
Mandal
Arabinda
A
Digilio
Laura
L
Klotz
Ken
K
Flickinger
Charles J
CJ
Herr
John C
JC
D054758:A Kinase Anchor Proteins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology; D013075:Sperm Capacitation / Q000502:physiology; D013082:Sperm Tail / Q000378:metabolism / Q000648:ultrastructure; D013091:Spermatogenesis / Q000235:genetics / Q000502:physiology; D013094:Spermatozoa / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D013997:Time Factors; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D054758:A Kinase Anchor Proteins; C485611:Akap3 protein, mouse; C485479:Akap4 protein, mouse; C486726:Cabyr protein, mouse; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; C404565:ROPN1 protein, human; D020741:rho GTP-Binding Proteins