Identification and characterization of receptors for riboflavin carrier protein in the chicken oocyte. Role of the phosphopeptide in mediating receptor interaction.
Riboflavin carrier protein (RCP) is a phosphoglycoprotein found in the egg and the serum of laying birds and other animals. We have investigated the binding of chicken RCP (cRCP) to membranes prepared from the whole chicken oocytes. RCP binding had an absolute requirement for calcium, with an affinity (Kd 10(-8) M) high enough to be physiologically relevant. Ligand blotting experiments using labeled RCP and vitellogenin, with proteins solubilized from oocyte membranes, indicated that RCP and vitellogenin bound specifically to three proteins of Mr 380, 260 and 110 kDa. Vitellogenin also bound to proteins of Mr 515 kDa and 97 kDa, similar in size to those identified by receptor associated protein of RAP. Reduced and carboxyamidated RCP inhibited the binding of 125I-labeled RCP to chicken oocyte membranes, but recombinant RCP expressed in E. coli, and dephosphorylated RCP, failed to interact with the receptors, indicating that post-translational modifications were necessary for ligand-receptor interaction. The purified phosphopeptide, prepared from tryptic digests of egg white RCP, was able to inhibit the binding of RCP to the receptor proteins, with an affinity comparable to native RCP indicating that the phosphopeptide sequence present in RCP serves as the focal point for RCP-receptor interactions.
Sooryanarayana
Center for Reproductive Biology and Molecular Endocrinology, Indian Institute of Science, Bangalore, India.
Sarkar
S
S
Adiga
P R
PR
Visweswariah
S S
SS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002645:Chickens; D004926:Escherichia coli / Q000235:genetics; D008562:Membrane Glycoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009865:Oocytes / Q000737:chemistry*; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism; D014819:Vitellogenins / Q000378:metabolism
D001798:Blood Proteins; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D010748:Phosphopeptides; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014819:Vitellogenins; C019079:riboflavin-binding protein; D014357:Trypsin; D002118:Calcium