Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity - PubMed (original) (raw)

Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity

J Gosling et al. Proc Natl Acad Sci U S A. 1997.

Abstract

The C-C chemokine receptor 5 (CCR5) plays a crucial role in facilitating the entry of macrophage-tropic strains of the HIV-1 into cells, but the mechanism of this phenomenon is completely unknown. To explore the role of CCR5-derived signal transduction in viral entry, we introduced mutations into two cytoplasmic domains of CCR5 involved in receptor-mediated function. Truncation of the terminal carboxyl-tail to eight amino acids or mutation of the highly conserved aspartate-arginine-tyrosine, or DRY, sequence in the second cytoplasmic loop of CCR5 effectively blocked chemokine-dependent activation of classic second messengers, intracellular calcium fluxes, and the cellular response of chemotaxis. In contrast, none of the mutations altered the ability of CCR5 to act as an HIV-1 coreceptor. We conclude that the initiation of signal transduction, the prototypic function of G protein coupled receptors, is not required for CCR5 to act as a coreceptor for HIV-1 entry into cells.

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Figures

Figure 1

CCR5 truncations and mutations. Wild-type CCR5 was truncated by introducing stop codons after Phe-320 (F320, CCR5Δ1) or Leu-308 (L308, CCR5Δ2). Mutations of the indicated amino acids in the second intracellular loop were introduced by PCR to functionally uncouple the receptor.

Figure 2

Signal transduction and HIV-1 coreceptor activity mediated by CCR5 and CCR5 truncation mutants. HEK-293T cells were transiently transfected with CCR5, CCR5Δ1, and CCR5Δ2. (A) Inositol phosphate (IP) release. A fold increase of 1.0 corresponds to no activation. Shown is one of three similar experiments. (B) Calcium mobilization in response to MIP-1β (100 nM). Transiently transfected cells were loaded with indo-1AM, and intracellular calcium levels were measured as described (24). (C) Inhibition of adenylyl cyclase induced by MIP-1β. Transiently transfected cells were incubated with forskolin in the presence of the indicated concentrations of MIP-1β, and cAMP levels were determined as described (24). (D) The HIV-1 coreceptor activity of each truncation mutant was calculated as a percentage of the activity of each wild-type CCR5. The activity of CCR5 was defined as 100%. HIV-1 coreceptor activity was determined as described (18) using Ba-L. HEK-293T cells were cotransfected with human CD4 and the indicated chemokine receptor. CCR2 was included as a negative control, and the coreceptor activity of wild-type CCR5 was set at 100%. (Bars = SEM; n = 4.)

Figure 3

Binding of MIP-1β to cells transfected with CCR5 and CCR5-GGAA. HEK-293T cells were transiently transfected with CCR5 (A) or CCR5-GGAA (B) and incubated with 125I-labeled MIP-1β (5 nM, CCR5; 50 nM, CCR5-GGAA), as described (19). Shown is the Scatchard analysis of competition binding experiments with unlabeled MIP-1β. Similar results were obtained with radiolabeled MIP-1α. One of three similar experiments is presented.

Figure 4

Signal transduction mediated by CCR5 and CCR5-GGAA in transiently transfected HEK-293T cells. (A) Inositol phosphate (IP) release from HEK-293T cells transfected with CCR5 or CCR5-GGAA in response to the indicated chemokines (100 nM). A fold increase of 1.0 corresponds to no activation. Shown is one of three similar experiments. Similar results were seen with up to 600 nM MIP-1α, MIP-1β, and RANTES (regulated on activation, normal T cell expressed and secreted). (B) Inhibition of adenylyl cyclase in transiently transfected cells was determined as described in Fig. 1. The dose-response curve was obtained with wild-type CCR5 and MIP-1β. No response was seen to the indicated concentrations of MIP-1α, MIP-1β, or RANTES in cells transfected with CCR5-GGAA. (Bars = SEM.)

Figure 5

CCR5-GGAA failed to mobilize intracellular calcium or induce chemotaxis in 300–19 pre-B cells stably transfected with CCR5 or CCR5-GGAA as described. (A) Calcium mobilization in response to MIP-1β (100 nM) was measured as described in Fig. 2. (B) Chemotaxis was determined as described.

Figure 6

HIV-1 coreceptor activity of CCR5 and CCR5-GGAA in HEK-293T cells transfected with CD4 and either CCR5 or CCR5-GGAA, as described in Fig. 2. HIV-1 coreceptor activity was determined for Ba-L. CCR5 activity was set at 100%. (Bars = SEM, n = 3.)

References

1. Feng Y, Broder C C, Kennedy P E, Berger E A. Science. 1996;272:872–877. - PubMed
1. Alkhatib G, Combadiere C, Broder C C, Feng Y, Kennedy P E, Murphy P M, Berger E A. Science. 1996;272:1955–1958. - PubMed
1. Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath P D, Wu L, Mackay C R, LaRosa G, Newman W, Gerard N, Gerard C, Sodroski J. Cell. 1996;85:1135–1148. - PubMed
1. Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, Di Marzio P, Marmon S, Sutton R E, Hill C M, Davis C B, Peiper S C, Schall T J, Littman D R, Landau N R. Nature (London) 1996;381:661–666. - PubMed
1. Dragic T, Litwin V, Allaway G P, Martin S R, Huang Y, Nagashima K A, Cayanan C, Maddon P J, Koup R A, Moore J P, Paxton W A. Nature (London) 1996;381:667–673. - PubMed

Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity - PubMed (original) (raw)