CXCR1 and CXCR2 activation and regulation

Role of aspartate 199 of the second extracellular loop of CXCR2 in CXCL8-mediated rapid receptor internalization

Mohd W. Nasser, Sandeep K. Raghuwanshi, Kimberly M. Malloy, Pavani Gangavarapu, Joong Youn Shim, Krishna Rajarathnam, Ricardo M. Richardson

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

CXCL8 (interleukin-8) interacts with two receptors, CXCR1 and CXCR2, to activate leukocytes. Upon activation, CXCR2 internalizes very rapidly relative to CXCR1 (∼90% versus ∼10% after 5 min). The C termini of the receptors have been shown to be necessary for internalization but are not sufficient to explain the distinct kinetics of down-regulation. To determine the structural determinant(s) that modulate receptor internalization, various chimeric and point mutant receptors were generated by progressively exchanging specific domains or amino acids between CXCR1 and CXCR2. The receptors were stably expressed in rat basophilic leukemia 2H3 cells and characterized for receptor binding, intracellular Ca2+ mobilization, phosphoinositide hydrolysis, phosphorylation, internalization, and MAPK activation. The data herein indicate that the second extracellular loop (2ECL) of the receptors is critical for the distinct rate of internalization. Replacing the 2ECL of CXCR2 with that of CXCR1 (B2ECLA) or Asp199 with its CXCR1 valine counterpart (BD199VA) delayed CXCR2 internalization similarly to CXCR1. Replacing Asp199 with Asn (BD199N) restored CXCR2 rapid internalization. Structure modeling of the 2ECL of the receptors also suggested that Asp199 plays a critical role in stabilizing and modulating CXCR2 rapid internalization relative to CXCR1. BD199N internalized rapidly but migrated as a single phosphorylated form like CXCR1 (∼75 kDa), whereas B2ECLA and BD199VA showed slow and fast migrating forms like CXCR2 (∼45 and ∼65 kDa, respectively) but internalized like CXCR1. These data further undermine the role of receptor oligomerization in CXCL8 receptor internalization. Like CXCR1, B D199VA also induced sustained ERK activation and cross-desensitized Ca2+ mobilization to CCR5 relative to BD199N and CXCR2. Altogether, the data suggest that the 2ECL of the CXCL8 receptors is important in modulating their distinct rate of down-regulation and thereby signal length and post-internalization activities.

Original languageEnglish (US)
Pages (from-to)6906-6915
Number of pages10
JournalJournal of Biological Chemistry
Volume282
Issue number9
DOIs
StatePublished - Mar 2 2007

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Aspartic Acid
Interleukin-8A Receptors
Interleukin-8B Receptors
Down-Regulation
Chemical activation
Valine
Phosphatidylinositols
Interleukin-8
Oligomerization
Leukemia
Phosphorylation
Hydrolysis
Leukocytes
Amino Acids
Rats
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

CXCR1 and CXCR2 activation and regulation : Role of aspartate 199 of the second extracellular loop of CXCR2 in CXCL8-mediated rapid receptor internalization. / Nasser, Mohd W.; Raghuwanshi, Sandeep K.; Malloy, Kimberly M.; Gangavarapu, Pavani; Shim, Joong Youn; Rajarathnam, Krishna; Richardson, Ricardo M.

In: Journal of Biological Chemistry, Vol. 282, No. 9, 02.03.2007, p. 6906-6915.

Research output: Contribution to journalArticle

Nasser, Mohd W. ; Raghuwanshi, Sandeep K. ; Malloy, Kimberly M. ; Gangavarapu, Pavani ; Shim, Joong Youn ; Rajarathnam, Krishna ; Richardson, Ricardo M. / CXCR1 and CXCR2 activation and regulation : Role of aspartate 199 of the second extracellular loop of CXCR2 in CXCL8-mediated rapid receptor internalization. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 9. pp. 6906-6915.
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abstract = "CXCL8 (interleukin-8) interacts with two receptors, CXCR1 and CXCR2, to activate leukocytes. Upon activation, CXCR2 internalizes very rapidly relative to CXCR1 (∼90{\%} versus ∼10{\%} after 5 min). The C termini of the receptors have been shown to be necessary for internalization but are not sufficient to explain the distinct kinetics of down-regulation. To determine the structural determinant(s) that modulate receptor internalization, various chimeric and point mutant receptors were generated by progressively exchanging specific domains or amino acids between CXCR1 and CXCR2. The receptors were stably expressed in rat basophilic leukemia 2H3 cells and characterized for receptor binding, intracellular Ca2+ mobilization, phosphoinositide hydrolysis, phosphorylation, internalization, and MAPK activation. The data herein indicate that the second extracellular loop (2ECL) of the receptors is critical for the distinct rate of internalization. Replacing the 2ECL of CXCR2 with that of CXCR1 (B2ECLA) or Asp199 with its CXCR1 valine counterpart (BD199VA) delayed CXCR2 internalization similarly to CXCR1. Replacing Asp199 with Asn (BD199N) restored CXCR2 rapid internalization. Structure modeling of the 2ECL of the receptors also suggested that Asp199 plays a critical role in stabilizing and modulating CXCR2 rapid internalization relative to CXCR1. BD199N internalized rapidly but migrated as a single phosphorylated form like CXCR1 (∼75 kDa), whereas B2ECLA and BD199VA showed slow and fast migrating forms like CXCR2 (∼45 and ∼65 kDa, respectively) but internalized like CXCR1. These data further undermine the role of receptor oligomerization in CXCL8 receptor internalization. Like CXCR1, B D199VA also induced sustained ERK activation and cross-desensitized Ca2+ mobilization to CCR5 relative to BD199N and CXCR2. Altogether, the data suggest that the 2ECL of the CXCL8 receptors is important in modulating their distinct rate of down-regulation and thereby signal length and post-internalization activities.",
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AU - Malloy, Kimberly M.

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AU - Richardson, Ricardo M.

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