TRANSFECTED CELLS EXPRESS MOSTLY THE INTRACELLULAR PRECURSOR OF THE LUTROPIN CHORIOGONADOTROPIN RECEPTOR BUT THIS PRECURSOR BINDS CHORIOGONADOTROPIN WITH HIGH-AFFINITY/
J. Fabritz et al., TRANSFECTED CELLS EXPRESS MOSTLY THE INTRACELLULAR PRECURSOR OF THE LUTROPIN CHORIOGONADOTROPIN RECEPTOR BUT THIS PRECURSOR BINDS CHORIOGONADOTROPIN WITH HIGH-AFFINITY/, Biochemistry, 37(2), 1998, pp. 664-672
Previous studies from several laboratories have shown that the cell su
rface rLHR is a 85-92 kDa protein synthesized from a 68-73 kDa intrace
llular precursor. While all investigators agree that the cell surface
rLHR binds hCG with high affinity, it is not clear if the intracellula
r precursor can also bind hCG. In order to directly determine if the i
ntracellular rLHR present in cells transfected with the wild-type rLHR
binds hCG with high affinity, we devised a method that selectively de
grades 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 f
rom proteolyzed cells was largely preserved. Measurements of the hCG b
inding affinity to intact cells or to detergent extracts prepared befo
re and after proteolysis display very similar or identical binding aff
inities. Since binding to nonproteolyzed intact cells, detergent extra
cts prepared from nonproteolyzed cells, or detergent extracts prepared
from proteolyzed cells occurs only to the 85-92 kDa rLHR, the 85-92 a
nd 68-73 MDa rLHR, and the 68-73 kDa rLHR, respectively, we conclude t
hat the cell surface rLHR and the intracellular rLHR bind hCG with the
same affinity. Quantitation of the relative abundance: of the cell su
rface and intracellular rLHR by immunological methods indicates that t
ransfected cells express mostly the intracellular precursor. A compari
son 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.