The efficiency of covalent labeling of the pancreatic cholecystokinin receptor using a battery of crosslinkable and photolabile probes.
Affinity labeling is a powerful method for biochemical characterization of hormone receptors, dependent on approximation of reactive groups on ligand and receptor. In this work, we have compared the efficiency of covalent labeling of the rat pancreatic cholecystokinin (CCK) receptor by decapeptide probes with differing photolabile moieties sited at their amino-terminus, mid-region, or carboxyl-terminus, or chemically crosslinkable via their amino-terminus. Each labeled the same M(r) = 85,000-95,000 plasma membrane glycoprotein with a protein core of M(r) = 42,000. Affinity labeling this band through the amino-terminus of the decapeptide, 125I-D-Tyr-Gly [(Nle28,31)CCK-26-33], was inefficient using bifunctional chemical reagents, m-maleimidobenzoyl-N-hydroxy-succinimide ester (0.07% of total incubated radioactivity, representing 0.4% of specifically bound counts) or disuccinimidyl suberate (0.02, 0.2%) or a photolabile carbene precursor (0.06, 0.2%). A benzophenone at this locus yielded more efficient labeling of this band (0.09, 11.8%), but high levels of nonspecific labeling. Probes attached through residues within the receptor-binding domain were particularly useful. Photolabile derivatives of phenylalanine at the carboxyl-terminus of this domain yielded better incorporation (4-nitro-Phe33: 0.23, 1.4%; 4-azido-Phe3: 0.67, 6.0%). A 6-nitro-Trp30 derivative in the middle of this domain gave similarly efficient labeling (0.08, 3.5%) despite being a less potent pancreatic secretagogue. These studies clearly demonstrate that the efficiency of covalent labeling of a receptor can be markedly affected by the nature and site of crosslinking chosen.
Klueppelberg
U G
UG
Gastroenterology Research Unit, Mayo Clinic and Foundation, Rochester, MN 55905.
Powers
S P
SP
Miller
L J
LJ
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D006031:Glycosylation; D008297:Male; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010179:Pancreas / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011949:Receptors, Cholecystokinin / Q000032:analysis*
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D015335:Molecular Probes; D011949:Receptors, Cholecystokinin