Laminin-333/α6β1 Integrin is a Non-basement Membrane Cell Adhesion Protein Complex at the Ectoplasmic Specialization of the Rat Testis

Abstract

Apical ectoplasmic specialization (ES) is a testis-specific Sertoli-germ cell actin-based adherens junction (AJ) type restricted to the interface between Sertoli cells and spermatids. Laminin γ3 was shown to be a putative binding partner of α6β1 integrin at the apical ES. However, the αand βchains that can constitute a functional laminin receptor for α6β1 integrin at the apical ES are unknown. Using RT-PCR and immunoblottings to survey all laminin α, βand γchains in cells of the seminiferous epithelium, it was noted that α2, α3, β1, β2, β3 and γ3 chains were found in germ cells, whereas α1, α2, β1, β2, γ1 and γ3 were in Sertoli cells, implicatingα3 and β3 are the plausible laminin chainsrestricted to germ cells that can be the bona fide partners of γ3. To verify this postulate, differ ent cDNA constructs based on the Domain I of α3, β3 and γ3 were subcloned into pET101/D-TOPO® vector. Recombinant proteins were expressed in E. coli and purified using Ni-columns. Monospecific polyclonal antibodies against the α3, β3 and γ3 chains wer e raised in rabbits. Studies by immunoblottings, immunohistochemistry and immunofluorescence microscopy using testes and Sertoli/germ cell cocultures demonstrated that laminin α3, β3 and γ3 chains of 165, 140, 146 kDa, respectively, were indeed restricted to germ cells at the apical ES and co-localized with each other and β1 integrin. The protein levels of these three laminin chains and β1 integrin were also induced when functional apical ES was established in Sertoli-germ cell cocultures. More important, using coimmunoprecipitation technique, an anti-laminin γ3 antibody also pulled out α3 and β3 and vice versa. In summary, laminin-333 is the functional non-basement membrane receptor of α6β1 integrin at the apical ES.