Biology subjects

Lamme, T. D.

Publications and source records attributed to Lamme, T. D..

4 recordsLinked to original sources

bioRxiv2026

CCL25 targeting by de novo protein binders differentially suppress CCR9 and ACKR4 activation and transform CCL25 into a biased agonist

Chemokines and their receptors mediate cell migration and coordinate immune responses, while dysregulation can lead to inflammation. Therapeutic modulation of the chemokine signaling axis has proven difficult. Most drug discovery efforts target the receptors, whereas natural regulatory mechanisms focus on the chemokines. Despite this insight, development of effective chemokine-directed modulators has remained elusive. Recent advances in de novo protein design offer an unprecedented opportunity to produce high-affinity binders that efficiently block protein-protein interactions. We implemented a computational workflow leveraging the BindCraft platform to generate miniprotein binders against CCL25, the chemokine ligand for the receptors CCR9 and ACKR4 and implicated in inflammatory bowel diseases. The unbiased development results in several miniproteins designed to block the receptor N-terminus from wrapping the chemokine and prevent productive engagement. Thus, these proteins suppress CCL25-mediated effector coupling and halt MOLT-4 lymphoblast migration. Another class of miniprotein, represented by VUP25111, is predicted to bind CCL25 along the chemokine {beta}1 strand and retained receptor binding. This complex inhibited arrestin recruitment to CCR9, but not to ACKR4, indicating receptor specificity. Additionally, G protein signaling through CCR9 was unimpeded by VUP25111, suggesting that the miniprotein biased the native balanced agonist towards G proteins. These results demonstrate the effectiveness of differentially targeting CCL25 to suppress CCR9 signaling and new tools to resolve the structural basis of chemokine receptor activation and bias.

biochemistry↗

bioRxiv2026

GPCR kinase 3 phosphorylates atypical chemokine receptor 4 independent of G proteins

Atypical chemokine receptors (ACKRs) indirectly mediate cell migration through chemokine scavenging, which generally requires phosphorylation by G protein-coupled receptor (GPCR) kinases (GRKs) to efficiently control chemokine levels. Despite not coupling G proteins, ACKR4 is preferentially modified by GRK3, a kinase dependent on active G protein subunits for membrane translocation and phosphorylation activity. Here we resolve the underlying mechanisms allowing ACKR4 to circumvent the G protein requirement for GRK3 function. Using live cell BRET assays, we confirm that ACKR4 is preferentially phosphorylated by the GRK2/3 kinase family and that both GRK recruitment and receptor phosphorylation occur in the absence of activated G proteins. Instead, the kinases are recruited directly by a unique acidic rich motif in the proximal receptor C-terminus which coordinates productive phosphorylation reactions. Mutations in this region severely attenuated kinase recruitment and phosphorylation. Productive phosphorylation reaction plays a substantial role in the G protein-independent mechanism and a kinase-dead GRK3 (KD-GRK3) has severely reduced recruitment to ACKR4. This was not observed for KD-GRK3 translocation to GPCRs that recruit the kinase in a G protein-dependent manner. Together, these findings suggest that ACKR4 directly coordinates GRK3 recruitment and phosphorylation, highlighting a uniquely evolved atypical mechanism to utilize GRK2/3 while bypassing G protein activation and thereby supporting efficient chemokine scavenging by the atypical receptor.

biochemistry↗

bioRxiv2024

Constitutive activity of an atypical chemokine receptor revealed by inverse agonistic nanobodies

Chemokine stimulation of atypical chemokine receptor 3 (ACKR3) does not activate G proteins but recruits arrestins. It is a chemokine scavenger that indirectly influences responses by restricting the availability of CXCL12, an agonist shared with the canonical receptor CXCR4. ACKR3 is upregulated in numerous disorders. Due to limited insights in chemokine-activated ACKR3 signaling, it is unclear how ACKR3 contributes to pathological phenotypes. One explanation may be that high constitutive activity of ACKR3 drives non-canonical signaling through a basal receptor state. Here we characterize the constitutive action of ACKR3 using novel inverse agonistic nanobodies to suppress basal activity. These new tools promote an inactive receptor conformation which decreased arrestin engagement and inhibited constitutive internalization. Basal, non-chemotactic, breast cancer cell motility was also suppressed, suggesting a role for ACKR3 in this process. The basal receptor activity in pathophysiology may provide a new therapeutic approach for targeting ACKR3.

biochemistry↗

bioRxiv2024

CCR9 signal termination is governed by an arrestin-independent phosphorylation mechanism

The chemokine receptor CCR9 coordinates immune cell migration from the thymus to the small intestine along gradients of CCL25. Receptor dysregulation is associated with a variety of inflammatory bowel diseases such as Crohns and ulcerative colitis, while aberrant CCR9 overexpression correlates with tumor metastasis. Despite being an attractive therapeutic target, attempts to clinically antagonize CCR9 have been unsuccessful. This highlights the need for a deeper understanding of its specific regulatory mechanisms and signaling pathways. CCR9 is a G protein-coupled receptor (GPCR) and activates Gi and Gq pathways. Unexpectedly, live-cell BRET assays reveal only limited G protein activation and signaling is rapidly terminated. Truncating the receptor C-terminus significantly enhanced G protein coupling, highlighting the regulatory role of this domain. Signal suppression was not due to canonical arrestin-coordinated desensitization. Rather, removal of GPCR kinase (GRK) phosphorylation led to sustained and robust G protein activation by CCR9. Using site-directed mutagenesis, we identified specific phosphorylation patterns that attenuate G protein coupling. Receptor internalization does not correlate with G protein activation capabilities. Instead, CCR9 phosphorylation appeared to directly destabilize the interaction of G protein heterotrimers with the receptor. This interference could lead to rapid loss of productive coupling and downstream signaling as phosphorylation would effectively render the receptor incapable of G protein coupling. An arrestin-independent, phosphorylation-driven deactivation mechanism could complement arrestin-dependent regulation of other GPCRs and have consequences for therapeutically targeting these receptors.

biochemistry↗