Identification of specific inhibin A-binding proteins on mouse Leydig (TM3) and Sertoli (TM4) cell lines

The binding of human inhibin A to cell surface binding proteins of mouse Le ydig (TM3) and Sertoli (TM4) cell lines was investigated. Scatchard analysi s 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 phl and 10,400 sites/cell) cel ls. Compared with inhibin A, inhibin B only partially competed [I-125]inhib in A binding (6-8%), whereas activin A competed weakly (<0.01%). Chemical c ross-linking of [I-125]inhibin A to both cell lines identified five [I-125] 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 dis placement from intact TM3 (ED50 = 97 <plus/minus> 32 pM) and TM4 (ED50 = 75 +/- 28 pM) cells, suggesting that all of these proteins are involved in th e high affinity inhibin A binding complex. Immunoprecipitation of iodinated inhibin A complexed to TM3 and TM4 cells with an antibody against human be taglycan 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 id entity and may represent the putative inhibin receptor complex.