Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin

Abstract

Pre-B-cell growth-stimulating factor/ stromal cell-derived factor 1 (PBSF/SDF-1) is a member of the CXC group of chemokines that is initially identified as a bone marrow stromal cell-derived factor and as a pre-B-cell stimulatory factor. Although most chemokines are thought to be inducible inflammatory mediators, PBSF/SDF-1 is essential for perinatal viability,. B lymphopoiesis, bone marrow myelopoiesis, and cardiac ventricular septal formation, and it has chemotactic activities on resting lymphocytes and monocytes. In this paper, we have isolated a cDNA that encodes a seven transmembrane-spanning-domain receptor, designated pre-B-cell-derived chemokine receptor (PB-CKR) from a murine pre-B-cell clone, DW34. The deduced amino acid sequence has 90% identity with that of a HUMSTSR/fusin, a human immunodeficiency virus 1 (HIV-1) entry coreceptor. However, the second extracellular region has lower identity (67%) compared with HUMSTSR/fusin. PB-CKR is expressed during embryo genesis and in many organs and T cells of adult mice. Murine PBSF/SDF-1 induced an increase in intracellular free Ca2+ in DW34 cells and PB-CKR-transfected Chinese hamster ovary (CHO) cells, suggesting that PB-CKR is a functional receptor for murine PBSF/SDF-1. Murine PBSF/ SDF-1 also induced Ca2+ influx in fusin-transfected CHO cells. On the other hand, considering previous results that HIV-1 does not enter murine T cells that expressed human CD4, PB-CKR may not support HIV-1 infection. Thus, PB-CKR will be an important tool for functional mapping of HIV-1 entry coreceptor fusin and for understanding the function of PBSF/SDF-1 further.

Figure 1

Deduced amino acid sequence and transmembrane model of PB-CKR. The transmembrane-spanning domains were identified by hydropathy analysis and homology to other G-protein-coupled receptors. The solid circles represent amino acid substitutions of fusin.

Figure 2

Intracellular Ca²⁺ response of DW34 cells and CHO cells transfected with PB-CKR in response to murine PBSF/SDF-1. Ligands were added at the time indicated by the arrow. Rise in [Ca²⁺]_i is represented by the increase in relative fluorescence. DW34 cells (A) and CHO cells transfected with a vector encoding the PB-CKR (B and E) and fusin (D) were challenged with murine PBSF/SDF-1 once (A, B, and D) or twice (E). CHO cells transfected with a vector encoding the human MCP-1 receptor were challenged with murine PBSF/SDF-1 and human MCP-1 (C).

Figure 3

Northern blot analysis of PB-CKR mRNA expression. Poly(A)⁺ RNA from murine tissues was size-fractionated on formaldehyde/agarose gel, transferred to a nylon filter, and hybridized with a radiolabeled fragment of PB-CKR cDNA. Sizes (kb) of relevant marker bands are shown at left.

Figure 4

Conservation of the PB-CKR gene in various species. A Southern blot filter that contained EcoRI-digested genomic DNA from human, monkey, rat, mouse, dog, cow, rabbit, and chicken was hybridized with a DNA probe from PB-CKR. Sizes (kb) of relevant marker bands are shown at left.