Stigmoid bodies contain type I receptor proteins SorLA/LR11 and sortilin: new perspectives on their function.

Stigmoid bodies (SBs) are structures in the cytoplasm of neurons. SBs are mostly found in the hypothalamic region of the rat and contain a protein called huntingtin-associated protein 1 (HAP1). In a recent publication, large cytoplasmic structures were shown to be immunoreactive for a type I receptor called SorLA/LR11. By light microscopic analysis, these structures appeared similar to SBs in size and in brain regional and subcellular localization. To determine whether these large puncta correspond to HAP1-containing SBs, we used antibodies specific to various domains of the apolipoprotein receptor LR11 to perform immunocytochemistry in rat and mouse brain tissue. Transfection studies using HeLa cells were conducted to demonstrate the specificity of the antibodies. We found that, in both species, antibodies to the domain II (or VSP10 for vacuolar sorting protein 10 domain) of LR11 immunoreact with large cytoplasmic structures. Co-localization immunolabeling experiments in rat brain tissue sections and in neuron cultures showed that these LR11-immunoreactive structures correspond to HAP1-positive SBs. Electron microscopy was performed in rat hypothalamus and further demonstrated the presence of LR11 in SBs and its co-localization with HAP1. LR11-containing SBs were most abundant in the hypothalamus but were also found in many brainstem nuclei, thalamus, and hippocampus. Our data also show that sortilin, another transmembrane protein containing a VPS10 domain, localizes to large cytoplasmic puncta and is found in LR11-positive and Hap1-positive SBs in hypothalamic neuron cultures.

SorLA, a member of the LDL receptor family, is expressed in the collecting duct of the murine kidney.

SorLA/LR11 is a recently identified member of the LDL receptor superfamily, which is broadly expressed in the nervous system, but also in some non-neuronal tissues. To investigate sorLA expression in more detail we generated sorLA-specific antibodies. The kidney-specific expression of sorLA was investigated by in situ hybridization, immunohistochemistry, and immunofluorescence of murine kidney sections. In situ hybridizations of embryonic and adult kidney sections revealed transcripts mainly in the renal medulla. This expression pattern correlated with sorLA immunostainings. By co-staining with various markers sorLA expression was found to be restricted to cortical and medullary collecting tubules. SorLA was detected in intracellular vesicular structures, but not in the plasma membrane. Vesicles were distributed throughout the cytoplasm of cortical collecting tubules, whereas a more apical localization was noted in the medullary part. This intracellular compartment partially overlapped with the trans-Golgi marker gamma-adaptin and the transferrin receptor, a marker for shuttling compartments, but not with the lysosomal marker lamp2. These results demonstrate a highly specific expression of sorLA in collecting tubules. SorLA overlaps with intracellular shuttling compartments, indicating a role of sorLA for intracellular trafficking in the kidney.

Characterization of the VPS10 domain of SorLA/LR11 as binding site for the neuropeptide HA.

The single transmembrane receptor sorLA/LR11 contains binding domains typical for the low-density lipoprotein receptors and a VPS10 domain which, in the related receptor sortilin, binds the neuropeptide neurotensin. SorLA is synthesized as a proreceptor which is processed to the mature form by a furin-like propeptidase. Endogenous sorLA and its hydra homologue HAB bind the neuropeptide head activator (HA). Transiently expressed partial sorLA constructs were investigated for ligand binding. We found that HA binds with nanomolar affinity to the VPS10 domain. The sorLA propeptide also bound to the VPS10 domain, whereas the receptor-associated protein RAP interacted both with the class A repeats and the VPS10 domain. The sorLA propeptide inhibited binding of HA to full-length sorLA and to the VPS10 domain. It also interfered with binding of HA to hydra HAB, which is taken as evidence for a highly conserved tertiary structure of the VPS10 domains of this receptor in hydra and mammals. The propeptide inhibited HA-stimulated mitosis and proliferation in the human neuroendocrine cell line BON and the neuronal precursor cell line NT2. We conclude that sorLA is necessary for HA signaling and function.