The efficiency of covalent labeling of the pancreatic cholecystokinin receptor using a battery of crosslinkable and photolabile probes.

U. G. Klueppelberg, S. P. Powers, Laurence J Miller

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1-11
Number of pages11
JournalReceptor
Volume1
Issue number1-2
StatePublished - Dec 1990

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Cross-Linking Reagents
Cholecystokinin Receptors
Membrane Glycoproteins
Phenylalanine
Radioactivity
Labeling
Esters
Cell Membrane
Hormones
Ligands
Proteins
Derivatives
Cell membranes
disuccinimidyl suberate
benzophenone
succinimide
cholecystokinin (26-33)
carbene
Crosslinking
Rats

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

Cite this

The efficiency of covalent labeling of the pancreatic cholecystokinin receptor using a battery of crosslinkable and photolabile probes. / Klueppelberg, U. G.; Powers, S. P.; Miller, Laurence J.

In: Receptor, Vol. 1, No. 1-2, 12.1990, p. 1-11.

Research output: Contribution to journalArticle

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abstract = "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.",
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