Regulation of Cysteinyl Leukotriene Type 1 Receptor Internalization and Signaling

• Published in: The Journal of biological chemistry Vol. 280; no. 10; pp. 8722 - 8732
• Main Authors: Naik, Snehal, Billington, Charlotte K., Pascual, Rodolfo M., Deshpande, Deepak A., Stefano, Frank P., Kohout, Trudy A., Eckman, Delrae M., Benovic, Jeffrey L., Penn, Raymond B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.03.2005; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Cell Line; Chlorocebus aethiops; COS Cells; Humans; Kidney; Kinetics; Leukotriene D4 - pharmacology; Membrane Proteins - genetics; Membrane Proteins - metabolism; Membrane Proteins - physiology; Models, Molecular; Molecular Sequence Data; Open Reading Frames; Protein Conformation; Protein Transport; Receptors, Leukotriene - genetics; Receptors, Leukotriene - metabolism; Receptors, Leukotriene - physiology; Signal Transduction; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M413014200

Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance, no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and found regulation of internalization and signaling of the CysLT1R to be unique among G protein-coupled receptors. Rapid and profound LTD4-stimulated internalization was observed for the wild type (WT) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, suggesting a region within amino acids 310–321 as critical to internalization. Although overexpression of WT arrestins significantly increased WT CysLT1R internalization, expression of dominant-negative arrestins had minimal effects, and WT CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin-2 and arrestin-3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacologic inhibition of protein kinase C (PKC) was shown to profoundly inhibit CysLT1R internalization while greatly increasing both phosphoinositide (PI) production and calcium mobilization stimulated by LTD4 yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313–316)A) reduced receptor internalization, increased PI production and calcium mobilization by LTD4, and significantly attenuated the effects of PKC inhibition. These findings characterized the CysLT1R as the first G protein-coupled receptor identified to date in which PKC is the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization.