Calcium-dependent binding of annexin 12 to phospholipid bilayers: stoichiometry and implications.

Annexins (ANXs) are a superfamily of proteins whose functional hallmark is Ca2+-dependent binding to anionic phospholipids. Their core domains are usually composed of a 4-fold repeat of a conserved amino acid sequence, with each repeat containing a type II Ca2+ binding site that is generally thought to mediate Ca2+-dependent binding to the membrane. We now report that ANX12 binding to phospholipid vesicles is highly cooperative with respect to Ca2+ concentration (Hill constant approximately 7), thereby suggesting that more than the four well-characterized type II Ca2+ binding sites are involved in phospholipid binding. Two independent approaches, a novel 45Ca2+ copelleting assay and isothermal titration calorimetry, indicate a stoichiometry of approximately 12 mol of Ca2+/mol of ANX12 for binding to phospholipid vesicles. On the basis of the "low-affinity" Ca2+-binding sites in a number of ANX X-ray crystal structures, we propose a model for ANX12 bilayer binding that involves three types of Ca2+ sites in each of the four repeats. In this model, there is a complementarity between the spacing of the ANX12 Ca2+ binding sites and the spacing of the phospholipid headgroups in bilayers. We tested the implications of the model by manipulating the physical state of vesicles composed of phospholipids with saturated acyl chains with temperature and measuring its influence on ANX12 binding. ANX12 bound to vesicles in a Ca2+-dependent manner when the vesicles were in the liquid crystal phase but not when the phospholipid was in the gel phase. Furthermore, ANX12 bound initially to fluid bilayers remained bound when cooled to 4 degrees C, a temperature that should induce the gel phase transition. Overall, these studies suggest that ANX12 is well suited to being a Ca2+ sensor for rapid all-or-none intercellular membrane-related events.

Inward rectifier potassium channel Kir2.2 is associated with synapse-associated protein SAP97.

The strong inwardly rectifying potassium channels Kir2.x are involved in maintenance and control of cell excitability. Recent studies reveal that the function and localization of ion channels are regulated by interactions with members of the membrane-associated guanylate kinase (MAGUK) protein family. To identify novel interacting MAGUK family members, we constructed GST-fusion proteins with the C termini of Kir2.1, Kir2.2 and Kir2.3. GST affinity-pulldown assays from solubilized rat cerebellum and heart membrane proteins revealed an interaction between all three Kir2.x C-terminal fusion proteins and the MAGUK protein synapse-associated protein 97 (SAP97). A truncated form of the C-terminal GST-Kir2.2 fusion protein indicated that the last three amino acids (S-E-I) are essential for association with SAP97. Affinity interactions using GST-fusion proteins containing the modular domains of SAP97 demonstrate that the second PSD-95/Dlg/ZO-1 (PDZ) domain is sufficient for interaction with Kir2.2. Coimmunoprecipitations demonstrated that endogenous Kir2.2 associates with SAP97 in rat cerebellum and heart. Additionally, phosphorylation of the Kir2.2 C terminus by protein kinase A inhibited the association with SAP97. In rat cardiac ventricular myocytes, Kir2.2 and SAP97 colocalized in striated bands corresponding to T-tubules. In rat cerebellum, Kir2.2 was present in a punctate pattern along SAP97-positive processes of Bergmann glia in the molecular layer, and colocalized with astrocytes and granule cells in the granule cell layer. These results identify a direct association of Kir2.1, Kir2.2 and Kir2.3 with the MAGUK family member SAP97 that may form part of a macromolecular signaling complex in many different tissues.

Annexins V and XII alter the properties of planar lipid bilayers seen by conductance probes.

Annexins are proteins that bind lipids in the presence of calcium. Though multiple functions have been proposed for annexins, there is no general agreement on what annexins do or how they do it. We have used the well-studied conductance probes nonactin, alamethicin, and tetraphenylborate to investigate how annexins alter the functional properties of planar lipid bilayers. We found that annexin XII reduces the nonactin-induced conductance to approximately 30% of its original value. Both negative lipid and approximately 30 microM Ca(2+) are required for the conductance reduction. The mutant annexin XIIs, E105K and E105K/K68A, do not reduce the nonactin conductance even though both bind to the membrane just as wild-type does. Thus, subtle changes in the interaction of annexins with the membrane seem to be important. Annexin V also reduces nonactin conductance in nearly the same manner as annexin XII. Pronase in the absence of annexin had no effect on the nonactin conductance. But when added to the side of the bilayer opposite that to which annexin was added, pronase increased the nonactin-induced conductance toward its pre-annexin value. Annexins also dramatically alter the conductance induced by a radically different probe, alamethicin. When added to the same side of the bilayer as alamethicin, annexin has virtually no effect, but when added trans to the alamethicin, annexin dramatically reduces the asymmetry of the I-V curve and greatly slows the kinetics of one branch of the curve without altering those of the other. Annexin also reduces the rate at which the hydrophobic anion, tetraphenylborate, crosses the bilayer. These results suggest that annexin greatly reduces the ability of small molecules to cross the membrane without altering the surface potential and that at least some fraction of the active annexin is accessible to pronase digestion from the opposite side of the membrane.

Mapping genes that control hormone-induced ovulation rate in mice.

The present study mapped quantitative trait loci (QTL) that control 6-fold genetic differences in hormone-induced ovulation rate (HIOR) between C57BL/6J (B6) (HIOR = 54) and A/J strain mice (HIOR = 9). (The gene name is Ovulation Rate Induced [ORI] QTL and the gene symbol is Oriq.) QTL linkage analysis was conducted on 167 (B6xA)xA backcross mice at 165 loci. Suggestive B6 ORI QTL that control the number of eggs in cumulus mapped, as follows, near: Cyp19 and D9Mit4 on chromosome (Chr) 9 (Oriq1); D2Mit433 on Chr2 (Oriq2); D6Mit316 on Chr6 (Oriq3); DXMit22 on ChrX (Oriq4) and were associated with a 2.7, 2.7, 2.6, and 4.2 egg increases in HIOR, respectively. Oriq3 was significant (LOD = 3.45) based on composite interval mapping. QTL linkage analysis of the number of eggs matured by endogenous gonadotropins and ovulated by eCG mapped a significant Oriq5 to Chr 10 and suggestive Oriq to Chr 6, 7, and X. These data provide the first molecular genetic markers for reproductive QTL that control major differences in ovarian responsiveness to gonadotropins. These and closely linked syntenic molecular markers will enable a more accurate prediction of ovarian responsiveness to gonadotropins and provide selection criteria for improving reproductive performance in diverse mammalian species.

Annexin XII forms calcium-dependent multimers in solution and on phospholipid bilayers: a chemical cross-linking study.

The annexins are a family of proteins that bind in a Ca2+-dependent manner to phospholipids that are preferentially located on the intracellular face of plasma membranes. Recent X-ray studies of hydra annexin XII showed that it crystallized as a homohexamer with an intermolecular Ca2+ binding site separate from the type II Ca2+-dependent phospholipid binding site. On the basis of this hexamer structure, a novel mechanism was proposed to explain how annexins interact with membranes. The first step toward evaluating this proposal is to determine whether the annexin XII hexamer exists when the protein is not in a crystalline form. We now report that annexin XII in solution can be cross-linked with dimethyl suberimidate into multimers with apparent Mr's corresponding to trimers and hexamers as determined by SDS--polyacrylamide gel electrophoresis--the trimer band may correspond to incompletely cross-linked hexamers. Multimer formation was dependent on Ca2+ and was enhanced when the protein first was bound to phospholipid vesicles. To evaluate the role of the intermolecular Ca2+ site in annexin XII hexamer formation, one of the residues used to coordinate Ca2+, glutamate 105, was replaced with lysine (E105K). In solution, the E105K mutation inhibited hexamer formation in the presence of moderate (3 mM) but not high (25 mM) Ca2+. No inhibition of E105K annexin XII hexamer formation was observed in the presence of phospholipid, thereby suggesting that either (i) other interactions are capable of stabilizing the hexamer when bound to bilayers or (ii) only trimers form on bilayers and the observed hexamer bands were due to cross-linking of closely packed trimers. In summary, this study shows for the first time that annexin XII can form hexamers in solution and implicates the intermolecular Ca2+ site in hexamer formation. This study also shows that multimers form on bilayers but does not clearly establish whether the multimers are trimers or hexamers.

Calcium-dependent binding of S100C to the N-terminal domain of annexin I.

The annexin family of proteins is characterized by a conserved core domain that binds to phospholipids in a Ca(2+)-dependent manner. Each annexin also has a structurally distinct N-terminal domain that may impart functional specificity. To search for cellular proteins that interact with the N-terminal domain of annexin I, we constructed a fusion protein consisting of glutathione S-transferase fused to amino acids 2-47 of human annexin I (GST-AINT; AINT = annexin I N-terminal). Extracts from metabolically labeled A431 cells contained a single protein (M(r) approximately 10,000) that bound to GST-AINT in a Ca(2+)-dependent manner. A synthetic peptide corresponding to amino acids 2-18 of annexin I inhibited the binding of the 10-kDa protein to GST-AINT with half-maximal inhibition occurring at approximately 15 microM peptide. In cellular extracts, endogenous annexin I and the 10-kDa protein associated in a reversible Ca(2+)-dependent manner. Experiments with other annexins and with N-terminal truncated forms of annexin I indicated that the 10-kDa protein bound specifically to a site within the first 12 amino acids of annexin I. The 10-kDa protein was purified from human placenta by hydrophobic and affinity chromatography. Amino acid sequence analysis indicated that the 10-kDa protein is the human homologue of S100C, a recently identified member of the S100 subfamily of EF-hand Ca(2+)-binding proteins.

Crystal structure of the annexin XII hexamer and implications for bilayer insertion.

Annexins are a family of calcium- and phospholipid-binding proteins implicated in a number of biological processes including membrane fusion and ion channel formation. The crystal structure of the annexin XII hexamer, refined at 2.8 A resolution, forms a concave disk with 3-2 symmetry, about 100 A in diameter and 70 A thick with a central hydrophilic pore. Six intermolecular Ca2+ ions are involved in hexamer formation. An additional 18 Ca2+ ions are located on the perimeter of the disk, accessible only from the side of the hexameric disk. On the basis of the hexamer structure we propose here a new mode of protein-phospholipid bilayer interaction that is distinct from the hydrophobic insertion of typical membrane proteins. This speculative model postulates the Ca(2+)-dependent insertion of the hydrophilic annexin XII hexamer into phospholipid bilayers with local reorientation of the bilayer phospholipids.