Abstract 19123: Arginine Vasopressin Receptor 1A and Alpha 1A-Adrenergic Receptor Form Heteromeric Complexes on Human Vascular Smooth Muscle Cells
Introduction: Alpha 1-adrenergic receptors (α1-AR) and arginine vasopressin receptors (AVPR) are essential for regulating vascular smooth muscle function. Numerous previous observations demonstrated cross-talk between these receptors, but the underlying mechanisms remain poorly understood. Recent evidence suggests formation of heteromeric receptor complexes between G protein-coupled receptors as a possible mechanism for receptor cross-talk.
Hypothesis: We hypothesize that α1A-AR and arginine vasopressin receptor 1A (AVPR1A) form heteromeric complexes on human vascular smooth muscle cells (hVSMC).
Methods: Proximity ligation assays (PLA) were utilized to visualize and quantify individual receptors and receptor-receptor interactions on hVSMC (ATCC PCS-100-012) at single molecule resolution. PLA signals were quantified using the Duolink Image Tool software. Co-immunoprecipitation was used to confirm PLA findings. The selectivity of all antibodies were confirmed with siRNA knockdown of the corresponding receptors. Data are presented as mean ± SEM from n=3 independent experiments with n=10 vision fields analyzed per condition and experiment. Data were analyzed with one-way ANOVA and Dunnett’s post-hoc correction. A 2-tailed p<0.05 was considered significant.
Results: The following PLA signals/cell were obtained for the detection of individual receptors: AVPR1A - 1830.01 ± 256.10, α1A-AR - 65.99 ± 12.85, α1B-AR - 371.80 ± 57.61, α1D-AR - 167.90 ± 41.46 (p<0.05 vs. negative control for all). Receptor-receptor interactions were quantified as follows: α1A-AR:AVPR1A - 58.82 ± 8.790, α1A-AR:α1B-AR - 48.99 ± 4.85, α1A-AR:α1D-AR - 22.40 ± 6.612 and α1B-AR:α1D-AR - 33.35 ± 4.850. The number of PLA signals for all receptor interactions except for α1A-AR:α1D-AR were significant vs. negative control (omission of minus strand secondary probe in PLA, 1.877 ± 0.449). The interaction between AVPR1A and α1A-AR could be confirmed in co-immunoprecipitation experiments after AVPR1A pull-down.
Conclusions: Our observations confirm previous findings from expression systems on heteromeric complexes among α1-AR subtypes and suggest that heteromeric complexes between AVPR1A and α1A-AR are constitutively expressed on native hVSMC.
Author Disclosures: L.J. Albee: None. H.M. LaPorte: None. M. Majetschak: None.
- © 2016 by American Heart Association, Inc.