Abstract:
Nine membrane bound mammalian isoforms of adenylyl cyclase (mACs)
convert ATP into cAMP, an important second messenger in signal transduction.
Previous data have supported the notion that mACs membrane anchors could be
regarded as orphan receptors for unknown ligands that could establish a new way of
regulating the activity of mACs. Herein, I describe the work been done attempting to
isolate and identify a ligand that can modulate the activity of mACs via binding to its
membrane domain.
In the first study, lipids -expected as ligands- were isolated from fetal bovine
serum at different pH values. Lipidomic analysis identified glycerophospholipids (GPL)
as major constituents. Surprisingly, 1-stearoyl-2-docosahexaenoyl-phosphatidic acid
(SDPA) enhanced Gsñ activation of mAC3. Examining the specificity of the fatty acyl
substituents and head group of phosphatidic acid demonstrated a notable specificity.
We also showed the GPLs’ capability to affect other mACs differently, indicating a
modest specificity. Further data suggested SDPA binding to a cytosolic site. SDPA
enhanced mAC activity in mouse brain cortical membranes indicating its physiological
importance. Collectively, this study identified GPLs as intracellular effectors of mACs,
settling a new way of regulating mAC activities, and opening the door to looking for
other paths of mACs regulation.
In the second study, extraction and fractionation of bovine lung tissue
identified heme b that attenuated Gsñ-stimulated activities of all mAC isoforms.
Likewise, heme b attenuated class III bacterial ACs with similar efficacy to mACs. In
addition, it decreased cAMP accumulation in HEK293 cells and attenuated Gsñstimulated
AC activities in brain cortical membranes. Data suggested its direct binding
to the catalytic dimer. The study adds a new facet to the distinct physiological and toxic
actions played by heme b and evokes the possible linkage between the second
messenger cAMP and pathological conditions where heme b levels are elevated.
In the last study, we identified aliphatic lipids as mACs potential ligands.
Initially, oleic acid enhanced mAC2, 3, 7, and 9 activities with distinct efficacies. In a
cellular context, it enhanced cAMP accumulation in HEK293-mAC3. Exploring the
ligand space for those mACs identified other stimulating fatty acids with remarkable
specificity. Further analysis revealed the attenuation of mAC1 and 4 by arachidonic
acid and mAC5 and 6 by anandamide. To prove the specific ligand interaction with
mAC membrane domains, we generated an mAC5TM-mAC3cat chimera which was not
affected by oleic acid and attenuated by anandamide. The study validates a novel
receptor role for mAC membrane anchors and establishes a new way of cAMP
regulation; an interplay between rapid solute and tonic lipid signaling.
Adenylyl cyclase