Five-transmembrane domains appear sufficient for a G protein-coupled receptor: Functional five-transmembrane domain chemokine receptors

Abstract

The putative seven-transmembrane (TM) domains have been the structural hallmark for the superfamily of heterotrimeric G protein-coupled receptors (GPCRs) that regulate a variety of cellular functions by mediating a large number of extracellular signals. Five-TM GPCR mutants of chemokine receptor CCR5 and CXCR4, the Nterminal segment of which connected directly to TM3 as a result of a deletion of TM1-2 and the first intracellular and extracellular loops, have been obtained in this study. Laser confocal microscopy and f low cytometry analysis revealed that these five-TM mutant GPCRs were expressed stably on the cell surface after transfection into human embryonic kidney 293 cells. The five-TM CCR5 and CXCR4 functioned as normal chemokine receptors in mediating chemokinestimulated chemotaxis, Ca 2؉ inf lux, and activation of pertussis toxin-sensitive G proteins. Like the wild-type GPCRs, the five-TM mutant receptors also underwent agonist-dependent internalization and desensitization and were subjected to regulation by GPCR kinases and arrestins. Our study indicates that five-TM domains, at least in the case of CCR5 and CXCR4, appear to meet the minimum structural requirements for a functional GPCR and suggests possible existence of functional five-TM GPCRs in nature during evolution.

Two alternatively spliced GPR39 transcripts in seabream: molecular cloning, genomic organization, and regulation of gene expression by metabolic signals

“…According to the global toggle switch model, activation of seven TM receptors requires an inward movement of TM6 and TM7 toward the inner face of TM3 (Schwartz et al 2006). Consistent with this view, it is interesting to note that truncated chemokine receptors with five TM motifs lacking TM1 and TM2 act as fully functional GPCRs (Ling et al 1999). Moreover, receptor mutation analysis of GHSR and GPR39 indicates that TM6 and TM7 are essential for the constitutive receptor activity, further supporting the hypothesis that lacking in TM6 and TM7 2, midbrain; 3, hindbrain; 4, medulla; 5, pituitary; 6, hypothalamus; 7, intestine; 8, stomach; 9, muscle; 10, liver; 11, testis; 12, ovary; 13, gill filament; 14, heart; 15, adipose tissue; 16, kidney; 17, spleen; 18, negative control. has led to the inability of the truncated receptors in activating the signaling events ).…”

Complex Transcription and Splicing of Odorant Receptor Genes

“…This N-terminally truncated OR would start like intact OR with an extracellular domain. For chemokine receptors, which are also G-protein-coupled 7-TM receptors, it has been demonstrated that the five distal TM regions may be sufficient for signal transduction (37). Because 10 of the HLAlinked OR genes comprise the respective splice site as well as the alternative start codon at position 79 (6), one may expect a biological function also for the truncated proteins.…”

β-Arrestin2 Is Critically Involved in CXCR4-mediated Chemotaxis, and This Is Mediated by Its Enhancement of p38 MAPK Activation

“…The anti-␤-arrestin polyclonal antibody was produced in our laboratory as described (27). Full-length CXCR4 and ␤-arrestin2 clones were generated as described (28). T140 is a generous gift from Dr. Stephen C. Peiper (Dept.…”

“…Stable clones were selected and maintained in G418 (800 g/ml). For transient transfection, HEK293 cells were transfected by using the calcium phosphate-DNA co-precipitation method as described previously (28).…”