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The endoplasmic reticulum (ER) forms a complex endomembrane network that reaches into the cellular compartments of a neuron, including dendritic spines. Recent work discloses that the spine ER is a dynamic structure that enters and leaves spines. While evidence exists that ER Ca2+ release is involved in synaptic plasticity, the role of spine ER morphology remains unknown. Combining a new 3D spine generator with 3D Ca2+ modeling, we addressed the relevance of ER positioning on spine-to-dendrite Ca2+ signaling. Our simulations, which account for Ca2+ exchange on the plasma membrane and ER, show that spine ER needs to be present in distinct morphological conformations in order to overcome a barrier between the spine and dendritic shaft. We demonstrate that RyR-carrying spine ER promotes spine-to-dendrite Ca2+ signals in a position-dependent manner. Our simulations indicate that RyR-carrying ER can initiate time-delayed Ca2+ reverberation, depending on the precise position of the spine ER. Upon spine growth, structural reorganization of the ER restores spine-to-dendrite Ca2+ communication, while maintaining aspects of Ca2+ homeostasis in the spine head. Our work emphasizes the relevance of precise positioning of RyR-containing spine ER in regulating the strength and timing of spine Ca2+ signaling, which could play an important role in tuning spine-to-dendrite Ca2+ communication and homeostasis.
Reversible phosphorylation plays important roles in G protein-coupled receptor signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. Using metabolic 32P labeling and phosphopeptide sequencing we provide a complete phosphorylation map of the human bradykinin B2 receptor in its native cellular environment. We identified three serine residues, Ser(339), Ser(346), and Ser(348), at the C-terminal tail as principal phosphorylation sites. Constitutive phosphorylation occurs at Ser(348), while ligand-induced phosphorylation is found at Ser(339) and Ser(346)/Ser(348) that could be executed by several G protein-coupled receptor kinases. In addition, we found a protein kinase C-dependent phosphorylation of Ser(346) that was mutually exclusive with the basal phosphorylation at Ser(348) and therefore may be implicated in differential regulation of B2 receptor activation. Functional analysis of receptor mutants revealed that a low phosphorylation stoichiometry is sufficient to initiate receptor sequestration while a clustered phosphorylation around Ser(346) is necessary for desensitization of the B2 receptor-induced phospholipase C activation. This was further supported by the specifically reduced Ser(346)/Ser(348) phosphorylation observed upon stimulation with a nondesensitizing B2 receptor agonist. The differential usage of clustered phosphoacceptor sites points to distinct roles of multiple kinases in controlling G protein-coupled receptor function.