Modulation of secretory functions in epithelia by adenovirus capsid proteins.

To evaluate the safety of adenovirus-derived capsid proteins for ocular gene delivery, we have investigated their effects on the morphology and function of the acinar epithelial cells of the lacrimal gland. These cells are responsible for basal and stimulated release of proteins and electrolytes into ocular fluid, a process essential in maintaining the health of the ocular surface. Acinar epithelial cells from rabbit lacrimal gland were exposed to one of two adenovirus serotype 5 capsid proteins, penton or knob (the carboxy-terminal fragment of the fiber capsid protein). Sustained (16-18 h) exposure to the penton at 20 microg/ml was associated with major changes in the organization of the regulated secretory pathway and cytoskeleton. These changes included an apparent loss of mature secretory vesicles enriched in rab3D around the apical lumen as well as a depletion of apical actin. The microtubule array in penton-treated acini also exhibited bundling and disorganization. None of these effects were elicited by exposure to knob protein. Penton treatment also caused a significant (p < or = 0.05) increase and decrease in basal and carbachol-stimulated release, respectively, of bulk protein. Competition studies showed that RGD peptide partially prevented the penton-induced changes in rab3D-enriched secretory vesicles and actin filaments. These findings suggest that the adenovirus penton protein compromises normal acinar secretory compartment organization and function and that these changes are due at least partly to penton-integrin interactions.

A novel rabconnectin-3-binding protein that directly binds a GDP/GTP exchange protein for Rab3A small G protein implicated in Ca(2+)-dependent exocytosis of neurotransmitter.

BACKGROUND: Rab3A, a member of the Rab3 small G protein family, regulates Ca2+-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP) and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). We have recently found a novel protein, named rabconnectin-3, which is co-immunoprecipitated with Rab3 GEP or GAP from the extract of the crude synaptic vesicle (CSV) fraction of rat brain. Rabconnectin-3 is abundantly expressed in the brain where it is associated with synaptic vesicles. We have found that two more proteins are co-immunoprecipitated with Rab3 GEP from the CSV fraction of rat brain. We attempted here to isolate and characterize one of them. RESULTS: We determined its partial amino acid sequence, cloned its cDNA from a human cDNA library, and determined its primary structure. The protein consisted of 1490 amino acids (aa) and showed a calculated molecular weight of 163808. The protein had 7 WD domains. The protein was abundantly expressed in the brain where it co-localized with rabconnectin-3 on synaptic vesicles. The protein formed a stable complex with rabconnectin-3. We named this protein rabconnectin-3beta and renamed rabconnectin-3 rabconnectin-3alpha. Rabconnectin-3beta, but not rabconnectin-3alpha, directly bound Rab3 GEP. Neither rabconnectin-3alpha nor -3beta directly bound Rab3 GAP. CONCLUSION: These results indicate that rabconnectin-3 consists of the alpha and beta subunits and binds directly Rab3 GEP through the beta subunit and indirectly Rab3 GAP through an unidentified molecule(s).

Rabconnectin-3, a novel protein that binds both GDP/GTP exchange protein and GTPase-activating protein for Rab3 small G protein family.

Rab3A, a member of the Rab3 small G protein family, regulates Ca(2+)-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP), and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). It remains unknown how or in which step of the multiple exocytosis steps these regulators are activated and inactivated. We isolated here a novel protein that was co-immunoprecipitated with Rab3 GEP and GAP by their respective antibodies from the crude synaptic vesicle fraction of rat brain. The protein, named rabconnectin-3, bound both Rab3 GEP and GAP. The cDNA of rabconnectin-3 was cloned from a human cDNA library and its primary structure was determined. Human rabconnectin-3 consisted of 3,036 amino acids and showed a calculated M(r) of 339,753. It had 12 WD domains. Tissue and subcellular distribution analyses in rat indicated that rabconnectin-3 was abundantly expressed in the brain where it was enriched in the synaptic vesicle fraction. Immunofluorescence and immunoelectron microscopy revealed that rabconnectin-3 was concentrated on synaptic vesicles at synapses. These results indicate that rabconnectin-3 serves as a scaffold molecule for both Rab3 GEP and GAP on synaptic vesicles.

The RIM/NIM family of neuronal C2 domain proteins. Interactions with Rab3 and a new class of Src homology 3 domain proteins.

RIM1 is a putative effector protein for Rab3s, synaptic GTP-binding proteins. RIM1 is localized close to the active zone at the synapse, where it interacts in a GTP-dependent manner with Rab3 located on synaptic vesicles. We now describe a second RIM protein, called RIM2, that is highly homologous to RIM1 and also expressed primarily in brain. Like RIM1, RIM2 contains an N-terminal zinc finger domain that binds to Rab3 as a function of GTP, a central PDZ domain, and two C-terminal C(2) domains that are separated by long alternatively spliced sequences. Unexpectedly, the 3'-end of the RIM2 gene produces an independent mRNA that encodes a smaller protein referred as NIM2. NIM2 is composed of a unique N-terminal sequence followed by the C-terminal part of RIM2. Data bank searches identified a third RIM/NIM-related gene, which encodes a NIM isoform referred to as NIM3; no RIM transcript from this gene was detected. To test if NIMs, like RIMs, may function in secretion, we investigated the effect of NIM3 on calcium-triggered exocytosis in PC12 cells. NIM3 induced a dramatic increase in calcium-evoked exocytosis (50%), with no significant effect on base-line release, suggesting that NIMs, like RIMs, regulate exocytosis The combination of conserved and variable sequences in RIMs and NIMs indicates that the individual domains of these proteins provide binding sites for interacting molecules during exocytosis, as shown for the zinc finger domain of RIM, which binds to GTP-bound Rab3s. To search for additional interacting proteins for RIMs, we employed yeast two-hybrid screens with the C-terminal half of RIM1. Two members of a new family of homologous brain proteins, referred to as RIM-binding proteins (RIM-BPs), were identified. RIM-BPs bind to RIM in yeast two-hybrid and GST pull-down assays, suggesting a specific interaction. In RIMs, the binding site for RIM-BPs consists of a conserved proline-rich sequence between the two C(2) domains, N-terminal to the beginning of NIMs. RIM-BPs are composed of multiple domains, including three fibronectin type III-domains and three Src homology 3 domains, of which the second Src homology 3 domain binds to RIMs. With the RIM-BPs, we have identified a partner for RIMs that may bind to RIMs at the synapse in addition to Rab3.