Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Matthias Majetschak, MD, Ph.D.

Committee Member

Jerome Breslin, Ph.D.

Committee Member

Craig Doupnik, Ph.D.

Committee Member

David Lominadze, Ph.D.

Keywords

ADRA1, AVPR1A, Chemotaxis, Receptor oligomerization

Abstract

Neuroendocrine regulation of the innate immune system is a well-documentedphenomenon. The underlying mechanisms, however, are not well understood. Here we show that at least 20 members of the human chemokine receptor (CR) family heteromerize with one or more members of the α1-adrenergic receptor (AR) family and that all CRs, apart from CXCR1, heteromerize with arginine vasopressin receptor 1A (AVPR1A) in recombinant systems. These heteromeric complexes are detectable in human monocytes and the monocytic leukemia cell line THP-1. Though currently technical limitations prevent the direct visualization of endogenously expressed higher-order hetero-oligomeric receptor complexes, we provide evidence through functional analysis and proximity-based techniques that CRs that heteromerize with both α1B/D-ARs and AVPR1A exist and function within higher-order hetero-oligomeric receptor complexes in cells. Further, these hetero-oligomeric receptor complexes appear to form dynamic and interdependent networks of protein-protein interactions (PPIs), through which α1B/D-AR and AVPR1A ligands modulate the efficacy of their CR partners to mediate chemotaxis.

Analysis of function studies showed heteromerization between CRs and α1B-AR is essential for the normal function of CR heteromerization partners. siRNA depletion and CRISPR-Cas9 knockout of α1B-AR significantly reduced chemotaxis mediated by CR heteromerization partners of α1B-AR. Similarly, α1D-AR and AVPR1A siRNA depletion as well as CRISPR-Cas9 knockout of AVPR1A significantly reduced chemotaxis mediated by CCR2 and CXCR4, CRs whose heteromerization with α1B-AR was shown to be dependent on α1D-AR and AVPR1A co-expression. Likewise, phenylephrine and phentolamine reduced CR:α1B-AR heteromerization propensity, inhibited CR heteromerization partner mediated chemotaxis in vitro, and prevented leukocyte infiltration mediated via CR heteromerization partners in a murine air pouch model. In contrast, heteromerization of AVPR1A with CCR1 reduces the efficacy of CCR1 to mediate chemotaxis. CCR1 mediated chemotaxis was significantly enhanced in THP-1_AVPR1AKO cells, and arginine vasopressin and conivaptan, which were shown to reduce CR:AVPR1A heteromerization propensity significantly enhanced CCR1 mediated chemotaxis in transwell migration assays. The IC50 of α1-AR ligands to inhibit chemotaxis mediated by CR partners of α1B/D-ARs, and the EC50 and IC50 of AVPR1A ligands to modulate the efficacy of CR partners of AVPR1A are in the range of physiologically and pharmacologically relevant concentrations, which implies functional relevance of our findings in health and disease processes. Further, these findings advise caution in the interpretation of functional consequences after 7TM receptor knockdown in experimental models. Alterations of the heteromerization patterns among other receptor partners may alter physiological and pathological responses, in particular in more complex systems, such as studies on the function of isolated organs or in in vivo experiments.

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