Transfected cells express mostly the intracellular precursor of the lutropin/choriogonadotropin receptor but this precursor binds choriogonadotropin with high affinity.
Previous studies from several laboratories have shown that the cell surface rLHR is a 85-92 kDa protein synthesized from a 68-73 kDa intracellular precursor. While all investigators agree that the cell surface rLHR binds hCG with high affinity, it is not clear if the intracellular precursor can also bind hCG. In order to directly determine if the intracellular rLHR present in cells transfected with the wild-type rLHR binds hCG with high affinity, we devised a method that selectively degrades the cell surface rLHR while preserving the intracellular rLHR. The binding of hCG to intact cells was completely lost following mild proteolysis of the cells, but binding to detergent extracts prepared from proteolyzed cells was largely preserved. Measurements of the hCG binding affinity to intact cells or to detergent extracts prepared before and after proteolysis display very similar or identical binding affinities. Since binding to nonproteolyzed intact cells, detergent extracts prepared from nonproteolyzed cells, or detergent extracts prepared from proteolyzed cells occurs only to the 85-92 kDa rLHR, the 85-92 and 68-73 kDa rLHR, and the 68-73 kDa rLHR, respectively, we conclude that the cell surface rLHR and the intracellular rLHR bind hCG with the same affinity. Quantitation of the relative abundance of the cell surface and intracellular rLHR by immunological methods indicates that transfected cells express mostly the intracellular precursor. A comparison of the binding capacity of control and proteolyzed cells with that of their detergent extracts indicates that hCG binding assays greatly underestimate the relative abundance of the intracellular rLHR.
Fabritz
J
J
Department of Pharmacology, The University of Iowa, Iowa City, Iowa, 52242-1109, USA.
Ryan
S
S
Ascoli
M
M
D002462:Cell Membrane / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011974:Receptors, LH / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D006063:Chorionic Gonadotropin; D011498:Protein Precursors; D011974:Receptors, LH; D011994:Recombinant Proteins