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Identification of Specific Inhibin A-Binding Proteins on Mouse Leydig (TM3) and Sertoli (TM4) Cell Lines¹

Prince Henry’s Institute of Medical Research, Clayton 3168, Victoria, Australia

Address all correspondence to: Dr. David M. Robertson, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, 3168 Victoria, Australia. E-mail: david.robertson{at}med.monash.edu.au

The binding of human inhibin A to cell surface binding proteins of mouse Leydig (TM3) and Sertoli (TM4) cell lines was investigated. Scatchard analysis identified two classes of inhibin A-binding sites on TM3 (K_d(1) = 85 pM and 4,160 sites/cell; K_d(2) = 520 pM and 12,500 sites/cell) and TM4 (K_d(1) = 61 pM and 2,620 sites/cell; K_d(2) = 520 pM and 10,400 sites/cell) cells. Compared with inhibin A, inhibin B only partially competed [¹²⁵I]inhibin A binding (6–8%), whereas activin A competed weakly (<0.01%). Chemical cross-linking of [¹²⁵I]inhibin A to both cell lines identified five [¹²⁵I]inhibin A binding complexes with apparent molecular masses of 70, 95, 145, 155, and more than 200 kDa. Inhibin A displacement of [¹²⁵I]inhibin A from each of these cross-linked species (ED₅₀ = 60–110 pM) closely resembled displacement from intact TM3 (ED₅₀ = 97 ± 32 pM) and TM4 (ED₅₀ = 75 ± 28 pM) cells, suggesting that all of these proteins are involved in the high affinity inhibin A binding complex. Immunoprecipitation of iodinated inhibin A complexed to TM3 and TM4 cells with an antibody against human betaglycan identified protein complexes of more than 200, 145, and 95 kDa. It is concluded that the high affinity binding complex for inhibin A found in these cell lines consists of betaglycan and several proteins of unknown identity and may represent the putative inhibin receptor complex.

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