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 find regulation of internalization and signaling of the CysLT1R to be unique among GPCRs. 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, and suggested a region within amino acids 309-321 as critical to internalization. Co-expression of arrestin2 or arrestin3 increased agonist-stimulated internalization of wt CysLT1R while inhibiting phosphoinositide (PI) production. However, co-expression of dominant negative arrestins minimally affected internalization, and wt CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin2 and arrestin3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacological inhibition of PKC profoundly inhibited CysLT1R internalization while greatly increasing PI production 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 and increased PI production by LTD4, and significantly attenuated the effects of PKC inhibition. Heterologous desensitization by PKC was also observed, as pretreatment with phorbol ester caused a small but significant reduction in PI production with subsequent LTD4 challenge in cells expressing wt CysLT1R but not CysLT1RS(313-316)A. These findings characterize the CysLT1R as the first GPCR identified to date in which PKC is the principal regulator of rapid agonist-dependent internalization and desensitization.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI059755-01
Application #
6771287
Study Section
Special Emphasis Panel (ZRG1-ALTX-4 (02))
Program Officer
Dong, Gang
Project Start
2004-03-15
Project End
2008-02-29
Budget Start
2004-03-15
Budget End
2005-02-28
Support Year
1
Fiscal Year
2004
Total Cost
$323,156
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
937727907
City
Winston-Salem
State
NC
Country
United States
Zip Code
27157
Billington, Charlotte K; Penn, Raymond B; Hall, Ian P (2017) ?2 Agonists. Handb Exp Pharmacol 237:23-40
Deshpande, Deepak A; Theriot, Barbara S; Penn, Raymond B et al. (2008) Beta-arrestins specifically constrain beta2-adrenergic receptor signaling and function in airway smooth muscle. FASEB J 22:2134-41
Penn, Raymond B (2008) Embracing emerging paradigms of G protein-coupled receptor agonism and signaling to address airway smooth muscle pathobiology in asthma. Naunyn Schmiedebergs Arch Pharmacol 378:149-69
Penn, Raymond B; Benovic, Jeffrey L (2008) Regulation of heterotrimeric G protein signaling in airway smooth muscle. Proc Am Thorac Soc 5:47-57
Deshpande, Deepak A; Pascual, Rodolfo M; Wang, Si-Wei et al. (2007) PKC-dependent regulation of the receptor locus dominates functional consequences of cysteinyl leukotriene type 1 receptor activation. FASEB J 21:2335-42
Naik, Snehal; Billington, Charlotte K; Pascual, Rodolfo M et al. (2005) Regulation of cysteinyl leukotriene type 1 receptor internalization and signaling. J Biol Chem 280:8722-32