Leutropin/beta-adrenergic receptor chimeras bind choriogonadotropin and adrenergic ligands but are not expressed at the cell surface.
In some G-protein-coupled receptors (e.g. beta-adrenergic receptor (beta 2 AR)), the ligand-binding pocket is contained within the hydrophobic transmembrane domain. In others (e.g. luteinizing hormone receptor (LHR)), the relative roles of the extracellular N-terminal domain and the transmembrane region in hormone binding are unknown. To study the roles of these domains, we prepared vectors encoding the rat LHR N-terminal domain alone (L- -), the LHR N-terminal domain fused to the transmembrane and C-terminal domains of the vesicular stomatitis virus-G protein (LVV), the LHR N-terminal domain fused to the transmembrane and C-terminal domains of the hamster beta 2 AR (LAA), and the beta 2 AR N-terminal domain fused to the transmembrane and C-terminal domains of the rat LHR (ALL). Membrane preparations obtained from COS-7 cells expressing the beta 2 AR or LAA bound the beta-adrenergic antagonist 125I-cyanopindolol with equal affinity, confirming the observation that the beta 2 AR transmembrane domain forms the hormone-binding site. Membranes from COS-7 cells transfected with LHR bound 125I-human choriomic gonadotropin (hCG). However, membranes from LAA-, L(- -)-, and LVV-transfected cells had low capacity to bind 125I-hCG unless they were solubilized with Triton X-100. The affinity of the detergent-solubilized receptors for 125I-hCG was similar to that of the LHR. We were unable to detect binding of 125I-hCG to ALL in the presence or absence of detergent. These observations suggest that, whereas the transmembrane region of the beta 2 AR is sufficient to bind adrenergic ligands, the N-terminal region of the LHR is required for binding of hCG. Although the N terminus of the LHR is sufficient to bind hCG, both the N terminus and the transmembrane domains of the LHR are required for receptor expression on the cell surface.
Moyle
W R
WR
Department of Obstetrics and Gynecology, Robert Wood Johnson (Rutgers) Medical School, Piscataway, New Jersey 08854.
Bernard
M P
MP
Myers
R V
RV
Marko
O M
OM
Strader
C D
CD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002678:Chimera; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D003338:Corpus Luteum / Q000378:metabolism; D006224:Cricetinae; D005260:Female; D020120:Iodocyanopindolol; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010869:Pindolol / Q000031:analogs & derivatives* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000235:genetics / Q000378:metabolism*; D011974:Receptors, LH / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics
D006063:Chorionic Gonadotropin; C028391:G protein, vesicular stomatitis virus; D008024:Ligands; D008562:Membrane Glycoproteins; D015345:Oligonucleotide Probes; D011943:Receptors, Adrenergic, beta; D011974:Receptors, LH; D014759:Viral Envelope Proteins; D020120:Iodocyanopindolol; D010869:Pindolol; D007545:Isoproterenol