Ligand-induced internalization of the type 1 cholecystokinin receptor independent of recognized signaling activity

Erin E. Cawston, Kaleeckal G. Harikumar, Laurence J Miller

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Receptor ligands, identified as antagonists, based on the absence of stimulation of signaling, can rarely stimulate receptor internalization. D-Tyr-Gly-[(Nle 28,31,DTrp 30) CCK-26-32]-2-phenylethyl ester (D-Trp-OPE) is such a ligand that binds to the cholecystokinin (CCK) receptor and stimulates internalization. Here, the molecular basis of this trafficking event is explored, with the assumption that ligand binding initiates conformational change, exposing an epitope to direct endocytosis. Ligandstimulated internalization was studied morphologically using fluorescent CCK and D-Trp-OPE. D-Trp-OPE occupation of Chinese hamster ovary cell receptors stimulated internalization into the same region as CCK. Arrestin-biased action was ruled out using morphological translocation of fluorescent arrestin 2 and arrestin 3, moving to the membrane in response to CCK, but not D-Trp-OPE. Possible roles of the carboxyl terminus were studied using truncated receptor constructs, eliminating the proline-rich distal tail, the serine/threoninerich midregion, and the remainder to the vicinal cysteines. None of these constructs disrupted D-Trp-OPE-stimulated internalization. Possible contributions of transmembrane segments were studied using competitive inhibition with peptides that also had no effect. Intracellular regions were studied with a similar strategy using coexpressing cell lines. Peptides corresponding to ends of each loop region were studied, with only the peptide at the carboxyl end of the third loop inhibiting D-Trp-OPE-stimulated internalization but having no effect on CCK-stimulated internalization. The region contributing to this effect was refined to peptide 309-323, located below the recognized G protein-association motif. While a receptor in which this segment was deleted did internalize in response to D-Trp-OPE, it exhibited abnormal ligand binding and did not signal in response to CCK, suggesting an abnormal conformation and possible mechanism of internalization distinct from that being studied. This interpretation was further supported by the inability of peptide 309-323 to inhibit its D-Trp-OPE-stimulated internalization. Thus the 309-323 region of the type 1 CCK receptor affects antagonist-stimulated internalization of this receptor, although its mechanism and interacting partner are not yet clear.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume302
Issue number3
DOIs
StatePublished - Feb 2012

Fingerprint

Cholecystokinin Receptors
Cholecystokinin
Ligands
Peptides
Arrestin
Amino Acid Motifs
tyrosyl-glycyl-(norleucyl(28.31)-tryptophyl(30))-cholecystokinin (26-32) phenylethyl ester
Endocytosis
Cricetulus
Occupations
GTP-Binding Proteins
Proline
Serine
Cysteine
Tail
Epitopes
Ovary
Esters
Cell Line
Membranes

Keywords

  • Antagonist
  • G protein-coupled receptors
  • Receptor internalization

ASJC Scopus subject areas

  • Cell Biology
  • Physiology

Cite this

Ligand-induced internalization of the type 1 cholecystokinin receptor independent of recognized signaling activity. / Cawston, Erin E.; Harikumar, Kaleeckal G.; Miller, Laurence J.

In: American Journal of Physiology - Cell Physiology, Vol. 302, No. 3, 02.2012.

Research output: Contribution to journalArticle

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AB - Receptor ligands, identified as antagonists, based on the absence of stimulation of signaling, can rarely stimulate receptor internalization. D-Tyr-Gly-[(Nle 28,31,DTrp 30) CCK-26-32]-2-phenylethyl ester (D-Trp-OPE) is such a ligand that binds to the cholecystokinin (CCK) receptor and stimulates internalization. Here, the molecular basis of this trafficking event is explored, with the assumption that ligand binding initiates conformational change, exposing an epitope to direct endocytosis. Ligandstimulated internalization was studied morphologically using fluorescent CCK and D-Trp-OPE. D-Trp-OPE occupation of Chinese hamster ovary cell receptors stimulated internalization into the same region as CCK. Arrestin-biased action was ruled out using morphological translocation of fluorescent arrestin 2 and arrestin 3, moving to the membrane in response to CCK, but not D-Trp-OPE. Possible roles of the carboxyl terminus were studied using truncated receptor constructs, eliminating the proline-rich distal tail, the serine/threoninerich midregion, and the remainder to the vicinal cysteines. None of these constructs disrupted D-Trp-OPE-stimulated internalization. Possible contributions of transmembrane segments were studied using competitive inhibition with peptides that also had no effect. Intracellular regions were studied with a similar strategy using coexpressing cell lines. Peptides corresponding to ends of each loop region were studied, with only the peptide at the carboxyl end of the third loop inhibiting D-Trp-OPE-stimulated internalization but having no effect on CCK-stimulated internalization. The region contributing to this effect was refined to peptide 309-323, located below the recognized G protein-association motif. While a receptor in which this segment was deleted did internalize in response to D-Trp-OPE, it exhibited abnormal ligand binding and did not signal in response to CCK, suggesting an abnormal conformation and possible mechanism of internalization distinct from that being studied. This interpretation was further supported by the inability of peptide 309-323 to inhibit its D-Trp-OPE-stimulated internalization. Thus the 309-323 region of the type 1 CCK receptor affects antagonist-stimulated internalization of this receptor, although its mechanism and interacting partner are not yet clear.

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