Human leptin receptor. Determination of disulfide structure and N-glycosylation sites of the extracellular domain.
The leptin receptor (OB-R) is a member of the class I cytokine receptor family and mediates the weight regulatory effects of its ligand through interaction with cytoplasmic kinases. The extracellular domain of this receptor is comprised of two immunoglobulin-like and cytokine-receptor homology domains each and type III fibronectin domains. The extracellular domain of human leptin receptor was expressed in and purified from Chinese hamster ovary cells and was found to contain extensive N-glycosylation (approximately 36% of the total protein). The purified protein had a molecular weight of approximately 145,000 and exhibited ligand binding ability as evidenced by formation of ligand-receptor complex, followed by chemical cross-linking. The determined disulfide motif of the soluble leptin receptor contained several distinct cystine knots as well as 10 free cysteines. The N-glycosylation analysis revealed that Asn624 of the WSXWS motif (residues 622-626) within the C-terminal cytokine receptor homology domain was glycosylated, indicating that this region is solvent-exposed. On the other hand, the N-terminal WSXWS motif was not glycosylated.
Haniu
M
M
Departments of Protein Structure, Protein Chemistry, and Immunology, Amgen Inc., Thousand Oaks, California 91320-1789, USA. mhaniu@amgen.com
Arakawa
T
T
Bures
E J
EJ
Young
Y
Y
Hui
J O
JO
Rohde
M F
MF
Welcher
A A
AA
Horan
T
T
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010449:Peptide Mapping; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; C515448:LEPR protein, human; D008024:Ligands; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D010434:Pepsin A