Abstract
The genes encoding two functional human interleukin-8 (IL-8) receptors have been identified by molecular cloning techniques and they are members of the rhodopsin G-protein coupled receptor (GCR) superfamily. We report the molecular cloning of two rat GCR genes (rat CXCR1-like and rat CXCR2) whose conceptualized amino acid sequences are approximately 70% identical to the human IL-8 A and B receptor subtypes. The murine GRO-like peptide, macrophage inflammatory peptide-2 (MIP-2), elevates intracellular calcium levels in HEK293 cells expressing the rat CXCR2 receptor. Southern blot analysis of restriction-digested rodent and human genomic DNAs indicate that rat CXCR1-like and CXCR2 are: 1) each single copy genes in the rat genome; 2) most closely related to the human IL-8 receptor genes; and 3) orthologous to two previously identified murine genes. CXCR2 mRNA is detected in adult rat lung, spleen, and neutrophils. CXCR1-like mRNA can be detected in adult rat lung, native rat macrophages, and a rat alveolar macrophage cell line (NR8383). These data identify the rat orthologs of the human IL-8 receptors, and describe cellular and tissue targets of rat C-X-C chemokine peptides.
Highlights
Chemoattractant cytokines are structurally related pro-inflammatory peptides (ϳ70 –90 amino acids in length) which elicit leukocyte migration and activation
A search of the GenBank revealed that rat CXCR1-like was most similar to human receptors for IL-8
Rat CXCR2 was related to these human C-X-C chemokine receptors
Summary
Materials—DNA modifying enzymes were purchased from Promega (Madison, WI) with the exception of DNA polymerase (Pfu) which was purchased from Stratagene (La Jolla, CA). [␣-32P]dCTP (3000 Ci/mmol) and [␣-33P]dATP (2000 Ci/mmol) were purchased from DuPont/NEN (Wilmington, DE). [␣-32P]UTP (3000 Ci/mmol) and [␣-35S]ATP (1000 Ci/mmol) were from Amersham. PCR products were subcloned to the SmaI site of pGEM7ZF(ϩ) (Promega) and subjected to DNA sequence analysis Isolation of Rat Genomic Clones—Approximately 1 ϫ 106 independent recombinants of a Sau3aI partially digested Wistar rat genomic DNA library (Lambda Dash II, Stratagene) were simultaneously screened with 32P-radiolabeled 0.6 (pCrec4) and 0.7 (pCrec8) kbp PCR products. The PCR product was restriction digested with XbaI, cloned to the same site of pCDM8 (Invitrogen), and subjected to DNA sequence analysis. The transferred DNAs were hybridized to either the 32P-labeled 1.0-kbp EcoRI fragment of the rat CXCR1-like gene or the 0.7-kbp PCR product (pCrec8) encoding part of rat CXCR2, using the method of Church and Gilbert [26]. The antisense probe for rat CXCR2 was made in a similar manner using a HindIII/XbaI restriction fragment of rat CXCR2
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