G protein–coupled receptor kinase 3 couples atypical chemokine receptor 4 independent of G proteins

Thomas D. Lamme; Isabel B. Sánchez Arroyo; Martine J. Smit; Christopher T. Schafer

doi:10.1016/j.molpha.2026.100129

G protein–coupled receptor kinase 3 couples atypical chemokine receptor 4 independent of G proteins

Thomas D. Lamme
, Isabel B. Sánchez Arroyo
, Martine J. Smit
, Christopher T. Schafer^*

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

Atypical chemokine receptors (ACKRs) indirectly mediate cell migration through chemokine scavenging, which generally requires phosphorylation by G protein–coupled receptor kinases (GRKs) to efficiently control chemokine levels. Despite not coupling to G proteins, ACKR4 is preferentially modified by GRK3, a kinase dependent on active G protein subunits for membrane translocation and phosphorylation activity. How ACKR4 circumvents G protein dependency to engage GRK3 is unclear. To resolve the underlying mechanism, we used live-cell bioluminescence resonance energy transfer assays to measure GRK3 and phosphorylation-dependent arrestin recruitment and tracked the impact of fluorescent chemokine uptake by flow cytometry. We demonstrate that ACKR4 engages arrestin preferentially downstream of GRK2/3 phosphorylation fully independent of G protein coordination. Instead, the kinases are recruited directly to the atypical receptor via a unique acidic-rich motif in the proximal receptor C terminus. Mutations in this region severely impaired kinase and arrestin recruitment, as well as chemokine scavenging. Productive phosphorylation also plays a substantial role in G protein-independent GRK3 translocation to ACKR4, and recruitment of kinase-dead GRK3 is severely impaired. Together, these findings suggest that ACKR4 directly coordinates GRK3 coupling, highlighting a uniquely evolved atypical mechanism to use GRK2/3 while bypassing G protein activation and thereby supporting efficient chemokine scavenging by the atypical receptor. Significance Statement: Cell migration and positioning is efficiently regulated by atypical chemokine receptors (ACKR) through chemokine scavenging, often upon GRK phosphorylation. GRK3 dominates the phosphorylation of ACKR4, despite ACKR4 not activating G proteins needed to promote the kinase activity. This study resolved that ACKR4 is directly modified by GRK3 without G protein involvement. Instead, specific acidic residues coordinate the phosphorylation reaction. While seemingly unique to ACKR4, similar mechanisms for GRK2/3 action may contribute to kinase modification of other atypical and canonical GPCRs.

Original language	English
Article number	100129
Pages (from-to)	1-15
Number of pages	15
Journal	Molecular Pharmacology
Volume	108
Issue number	5-6
Early online date	12 May 2026
DOIs	https://doi.org/10.1016/j.molpha.2026.100129
Publication status	Published - Jun 2026

Bibliographical note

Publisher Copyright:
© 2026 The Author(s). Published by Elsevier Inc. on behalf of American Society for Pharmacology and Experimental Therapeutics. This is an open access article under the CC BY license. http://creativecommons.org/licenses/by/4.0/

Funding

We thank C. Hoffmann (Friedrich-Schiller-Universität), N. Heveker (Université de Montréal), N. Lambert (Augusta University), A. Inoue (Tohoku University), D. Legler (Biotechnology Institute Thurgau), and M. Bouvier (Université de Montréal) for the BRET constructs and cell lines used in this study.Financial supportThis publication is part of the TRANSLATION project with file number OCENW.M.24.006, which is partly financed by theDutch Research Council(NWO) [grant ID:https://doi.org/10.61686/YKUZU08217].Data availabilityThe authors declare that all data supporting the findings of this study are available within the paper and its supplemental data.CRediT authorship contribution statementThomas Lamme: Conceptualization, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – Original Draft, Writing – Review and Editing. Isabel Sánchez Arroyo: Investigation, Writing – Review and Editing. Martine J. Smit: Funding acquisition, Resources, Writing – Review and Editing. Christopher T. Schafer: Conceptualization, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Supervision, Validation, Visualization, Writing – Original Draft, Writing – Review and Editing.

Funders
Université de Montréal
Augusta University
Research Council, Rutgers, The State University of New Jersey
NWO

Keywords

ACKR4
Atypical Chemokine Receptor
Chemokine
Chemokine receptor
GPCR
GRK

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title = "G protein–coupled receptor kinase 3 couples atypical chemokine receptor 4 independent of G proteins",

abstract = "Atypical chemokine receptors (ACKRs) indirectly mediate cell migration through chemokine scavenging, which generally requires phosphorylation by G protein–coupled receptor kinases (GRKs) to efficiently control chemokine levels. Despite not coupling to G proteins, ACKR4 is preferentially modified by GRK3, a kinase dependent on active G protein subunits for membrane translocation and phosphorylation activity. How ACKR4 circumvents G protein dependency to engage GRK3 is unclear. To resolve the underlying mechanism, we used live-cell bioluminescence resonance energy transfer assays to measure GRK3 and phosphorylation-dependent arrestin recruitment and tracked the impact of fluorescent chemokine uptake by flow cytometry. We demonstrate that ACKR4 engages arrestin preferentially downstream of GRK2/3 phosphorylation fully independent of G protein coordination. Instead, the kinases are recruited directly to the atypical receptor via a unique acidic-rich motif in the proximal receptor C terminus. Mutations in this region severely impaired kinase and arrestin recruitment, as well as chemokine scavenging. Productive phosphorylation also plays a substantial role in G protein-independent GRK3 translocation to ACKR4, and recruitment of kinase-dead GRK3 is severely impaired. Together, these findings suggest that ACKR4 directly coordinates GRK3 coupling, highlighting a uniquely evolved atypical mechanism to use GRK2/3 while bypassing G protein activation and thereby supporting efficient chemokine scavenging by the atypical receptor. Significance Statement: Cell migration and positioning is efficiently regulated by atypical chemokine receptors (ACKR) through chemokine scavenging, often upon GRK phosphorylation. GRK3 dominates the phosphorylation of ACKR4, despite ACKR4 not activating G proteins needed to promote the kinase activity. This study resolved that ACKR4 is directly modified by GRK3 without G protein involvement. Instead, specific acidic residues coordinate the phosphorylation reaction. While seemingly unique to ACKR4, similar mechanisms for GRK2/3 action may contribute to kinase modification of other atypical and canonical GPCRs.",

keywords = "ACKR4, Atypical Chemokine Receptor, Chemokine, Chemokine receptor, GPCR, GRK",

author = "Lamme, \{Thomas D.\} and \{S{\'a}nchez Arroyo\}, \{Isabel B.\} and Smit, \{Martine J.\} and Schafer, \{Christopher T.\}",

note = "Publisher Copyright: {\textcopyright} 2026 The Author(s). Published by Elsevier Inc. on behalf of American Society for Pharmacology and Experimental Therapeutics. This is an open access article under the CC BY license. http://creativecommons.org/licenses/by/4.0/",

year = "2026",

month = jun,

doi = "10.1016/j.molpha.2026.100129",

language = "English",

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AU - Lamme, Thomas D.

AU - Sánchez Arroyo, Isabel B.

AU - Smit, Martine J.

AU - Schafer, Christopher T.

N1 - Publisher Copyright: © 2026 The Author(s). Published by Elsevier Inc. on behalf of American Society for Pharmacology and Experimental Therapeutics. This is an open access article under the CC BY license. http://creativecommons.org/licenses/by/4.0/

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N2 - Atypical chemokine receptors (ACKRs) indirectly mediate cell migration through chemokine scavenging, which generally requires phosphorylation by G protein–coupled receptor kinases (GRKs) to efficiently control chemokine levels. Despite not coupling to G proteins, ACKR4 is preferentially modified by GRK3, a kinase dependent on active G protein subunits for membrane translocation and phosphorylation activity. How ACKR4 circumvents G protein dependency to engage GRK3 is unclear. To resolve the underlying mechanism, we used live-cell bioluminescence resonance energy transfer assays to measure GRK3 and phosphorylation-dependent arrestin recruitment and tracked the impact of fluorescent chemokine uptake by flow cytometry. We demonstrate that ACKR4 engages arrestin preferentially downstream of GRK2/3 phosphorylation fully independent of G protein coordination. Instead, the kinases are recruited directly to the atypical receptor via a unique acidic-rich motif in the proximal receptor C terminus. Mutations in this region severely impaired kinase and arrestin recruitment, as well as chemokine scavenging. Productive phosphorylation also plays a substantial role in G protein-independent GRK3 translocation to ACKR4, and recruitment of kinase-dead GRK3 is severely impaired. Together, these findings suggest that ACKR4 directly coordinates GRK3 coupling, highlighting a uniquely evolved atypical mechanism to use GRK2/3 while bypassing G protein activation and thereby supporting efficient chemokine scavenging by the atypical receptor. Significance Statement: Cell migration and positioning is efficiently regulated by atypical chemokine receptors (ACKR) through chemokine scavenging, often upon GRK phosphorylation. GRK3 dominates the phosphorylation of ACKR4, despite ACKR4 not activating G proteins needed to promote the kinase activity. This study resolved that ACKR4 is directly modified by GRK3 without G protein involvement. Instead, specific acidic residues coordinate the phosphorylation reaction. While seemingly unique to ACKR4, similar mechanisms for GRK2/3 action may contribute to kinase modification of other atypical and canonical GPCRs.

AB - Atypical chemokine receptors (ACKRs) indirectly mediate cell migration through chemokine scavenging, which generally requires phosphorylation by G protein–coupled receptor kinases (GRKs) to efficiently control chemokine levels. Despite not coupling to G proteins, ACKR4 is preferentially modified by GRK3, a kinase dependent on active G protein subunits for membrane translocation and phosphorylation activity. How ACKR4 circumvents G protein dependency to engage GRK3 is unclear. To resolve the underlying mechanism, we used live-cell bioluminescence resonance energy transfer assays to measure GRK3 and phosphorylation-dependent arrestin recruitment and tracked the impact of fluorescent chemokine uptake by flow cytometry. We demonstrate that ACKR4 engages arrestin preferentially downstream of GRK2/3 phosphorylation fully independent of G protein coordination. Instead, the kinases are recruited directly to the atypical receptor via a unique acidic-rich motif in the proximal receptor C terminus. Mutations in this region severely impaired kinase and arrestin recruitment, as well as chemokine scavenging. Productive phosphorylation also plays a substantial role in G protein-independent GRK3 translocation to ACKR4, and recruitment of kinase-dead GRK3 is severely impaired. Together, these findings suggest that ACKR4 directly coordinates GRK3 coupling, highlighting a uniquely evolved atypical mechanism to use GRK2/3 while bypassing G protein activation and thereby supporting efficient chemokine scavenging by the atypical receptor. Significance Statement: Cell migration and positioning is efficiently regulated by atypical chemokine receptors (ACKR) through chemokine scavenging, often upon GRK phosphorylation. GRK3 dominates the phosphorylation of ACKR4, despite ACKR4 not activating G proteins needed to promote the kinase activity. This study resolved that ACKR4 is directly modified by GRK3 without G protein involvement. Instead, specific acidic residues coordinate the phosphorylation reaction. While seemingly unique to ACKR4, similar mechanisms for GRK2/3 action may contribute to kinase modification of other atypical and canonical GPCRs.

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ER -

G protein–coupled receptor kinase 3 couples atypical chemokine receptor 4 independent of G proteins

Abstract

Bibliographical note

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Keywords

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