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 N-terminal 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 flow 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 chemokine-stimulated chemotaxis, Ca2+ influx, 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.

Figure 1

(A) Amino acid sequence alignment of the putative TM1–3 region of the wild-type and the five-TM chemokine receptors CCR5 (wCCR5 and mCCR5) and CXCR4 (wCXCR4 and mCXCR4). The deleted residues in the mutants are presented by dashes. (B) Schematic two-dimensional model of the five-TM mutant chemokine receptors. The arrow indicates the connecting point of the N terminus to TM3.

Figure 2

Expression and internalization of the five-TM chemokine receptors. (A) The expression of wCCR5 (lane 1), mCCR5 (lane 2), wCXCR4 (lane 3), and mCXCR4 (lane 4) in transiently transfected HEK293 cells was detected by immunoprecipitation and Western blotting with 12CA5. (B–E) Cells transiently transfected with wCCR5 (B), mCCR5 (C), wCXCR4 (D), and mCXCR4 (E) were incubated without (Left) or with (Right) 10 nM agonist (RANTES for CCR5 and SDF-1 for CXCR4) for 30 min, and internalization of receptors from the cell surface was analyzed by laser confocal fluorescence microscopy using 12CA5 and FITC-conjugated anti-mouse IgG. (F) Similarly, internalization of the wild-type and five-TM mutant CCR5 or CXCR also was determined by using flow cytometry after incubation with or without (control) 10 nM chemokine. Untransfected cells or mock-transfected cells showed negative staining under the same conditions (not shown). Pictures shown in A–E are representative of two separate experiments. The data in F indicate averages and error ranges of two independent determinations in duplicate. ∗∗, P < 0.01 compared with unpretreated controls.

Figure 3

The five-TM chemokine receptor-mediated cellular responses. HEK293 cells transiently (A, B, and D) or stably (C) expressing the wild-type or mutant chemokine receptors were treated with indicated amounts (A and B), 1 nM (C), or 10 nM (D) of chemokines. The chemokine-stimulated G protein activation (A), adenylyl cyclase inhibition (B), cell migration (C), and Ca²⁺ influx (D) were measured. The EC₅₀ values of the chemokine-induced GTPγS binding were estimated: wCCR5, 0.15 nM; mCCR5, 0.18 nM; wCXCR4, 2.6 nM; and mCXCR4, 4.6 nM. The basal GTPγS binding values were in the range of 1.11 ± 0.04 nmol/mg protein. The EC₅₀ values for cyclase inactivation were 30 pM for wCCR5, 22 pM for mCCR5, 200 pM for wCXCR4, and 550 pM for mCXCR4. The untreated forskolin-stimulated cAMP levels were in the range of 129 ± 11 pmol/mg protein. The migration number of the untreated cells was 12.6 ± 0.3. All data are means ± SE of three independent experiments performed in duplicate.

Figure 4

Functional coupling of the five-TM chemokine receptors to PTX-sensitive G proteins. HEK293 cells were transiently transfected the wild-type or mutant chemokine receptors and pretreated with or without (control) 100 ng/ml PTX for 24 hr (A). The cells were transfected with the indicated chemokine receptor alone (control) or cotransfected with Giα2 (B) or Goα1 (C). The basal GTPγS binding was 1.08 ± 0.05 nmol/mg protein. Data presented are averages and error ranges of two separate experiments performed in duplicate. ∗∗, P < 0.01 compared with controls.

Figure 5

Desensitization and functional interaction of the five-TM chemokine receptor with receptor kinases and arrestins. HEK293 cells transiently expressing the wild-type or mutant chemokine receptors were pretreated with PBS (as control), 10 nM indicated chemokine (A), or 1 μM PMA (B) for 15 min at 37°C. After rinsing with PBS, the cells were challenged with 10 nM chemokine (RANTES for CCR5 and SDF-1α for CXCR4) and forskolin-stimulated cAMP formation was determined. The cells were transiently transfected with the wild-type or mutant CCR5 (C) or CXCR4 receptors (D) alone (control) or cotransfected with GRK2, GRK5, β-arrestin 1, or β-arrestin 2 as indicated. The inhibition of the cAMP formulation induced by 10 nM chemokines (RANTES for CCR5 and SDF-1α for CXCR4) was determined. (A) The untreated forskolin-stimulated cAMP levels were in the range of 123 ± 12 pmol/mg protein. Data presented are averages and error ranges of two separate experiments performed in duplicate. ∗∗, P < 0.01 compared with controls.