Calcium binding to calmodulin leads to an N-terminal shift in its binding site on the ryanodine Receptor.

The skeletal muscle calcium release channel, ryanodine receptor, is activated by calcium-free calmodulin and inhibited by calcium-bound calmodulin. Previous biochemical studies from our laboratory have shown that calcium-free calmodulin and calcium bound calmodulin protect sites at amino acids 3630 and 3637 from trypsin cleavage (Moore, C. P., Rodney, G., Zhang, J. Z., Santacruz-Toloza, L., Strasburg, G., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). We now demonstrate that both calcium-free calmodulin and calcium-bound calmodulin bind with nanomolar affinity to a synthetic peptide matching amino acids 3614-3643 of the ryanodine receptor. Deletion of the last nine amino acids (3635-3643) destroys the ability of the peptide to bind calcium-free calmodulin, but not calcium-bound calmodulin. We propose a novel mechanism for calmodulin's interaction with a target protein. Our data suggest that the binding sites for calcium-free calmodulin and calcium-bound calmodulin are overlapping and, when calcium binds to calmodulin, the calmodulin molecule shifts to a more N-terminal location on the ryanodine receptor converting it from an activator to an inhibitor of the channel. This region of the ryanodine receptor has previously been identified as a site of intersubunit contact, suggesting the possibility that calmodulin regulates ryanodine receptor activity by regulating subunit-subunit interactions.

Determinants for calmodulin binding on voltage-dependent Ca2+ channels.

Calmodulin, bound to the alpha(1) subunit of the cardiac L-type calcium channel, is required for calcium-dependent inactivation of this channel. Several laboratories have suggested that the site of interaction of calmodulin with the channel is an IQ-like motif in the carboxyl-terminal region of the alpha(1) subunit. Mutations in this IQ motif are linked to L-type Ca(2+) current (I(Ca)) facilitation and inactivation. IQ peptides from L, P/Q, N, and R channels all bind Ca(2+)calmodulin but not Ca(2+)-free calmodulin. Another peptide representing a carboxyl-terminal sequence found only in L-type channels (designated the CB domain) binds Ca(2+)calmodulin and enhances Ca(2+)-dependent I(Ca) facilitation in cardiac myocytes, suggesting the CB domain is functionally important. Calmodulin blocks the binding of an antibody specific for the CB sequence to the skeletal muscle L-type Ca(2+) channel, suggesting that this is a calmodulin binding site on the intact protein. The binding of the IQ and CB peptides to calmodulin appears to be competitive, signifying that the two sequences represent either independent or alternative binding sites for calmodulin rather than both sequences contributing to a single binding site.

Regulation of RYR1 activity by Ca(2+) and calmodulin.

The skeletal muscle calcium release channel (RYR1) is a Ca(2+)-binding protein that is regulated by another Ca(2+)-binding protein, calmodulin. The functional consequences of calmodulin's interaction with RYR1 are dependent on Ca(2+) concentration. At nanomolar Ca(2+) concentrations, calmodulin is an activator, but at micromolar Ca(2+) concentrations, calmodulin is an inhibitor of RYR1. This raises the question of whether the Ca(2+)-dependent effects of calmodulin on RYR1 function are due to Ca(2+) binding to calmodulin, RYR1, or both. To distinguish the effects of Ca(2+) binding to calmodulin from those of Ca(2+) binding to RYR1, a mutant calmodulin that cannot bind Ca(2+) was used to evaluate the effects of Ca(2+)-free calmodulin on Ca(2+)-bound RYR1. We demonstrate that Ca(2+)-free calmodulin enhances the affinity of RYR1 for Ca(2+) while Ca(2+) binding to calmodulin converts calmodulin from an activator to an inhibitor. Furthermore, Ca(2+) binding to RYR1 enhances its affinity for both Ca(2+)-free and Ca(2+)-bound calmodulin.

The survival motor neuron protein interacts with the transactivator FUSE binding protein from human fetal brain.

To identify interacting proteins of survival motor neuron (SMN) in neurons, a fetal human brain cDNA library was screened using the yeast two-hybrid system. One identified group of SMN interacting clones encoded the DNA transactivator FUSE binding protein (FBP). FBP overexpressed in HEK293 cells or endogenously expressed in fetal and adult mouse brain bound specifically in vitro to recombinant SMN protein. Furthermore, an anti-FBP antibody specifically co-immunoprecipitated SMN when both proteins were overexpressed in HEK293 cells. These results demonstrate that FBP is a novel interacting partner of SMN and suggests a possible role for SMN in neuronal gene expression.

Differential subcellular localization of the survival motor neuron protein in spinal cord and skeletal muscle.

To compare the expression pattern of the survival motor neuron (SMN) protein in spinal cord and skeletal muscle, we generated a sheep polyclonal antibody against a bacterially expressed human SMN-fusion protein. On Western blots, the affinity purified anti-SMN antibody recognized a approximately 38 kDa protein band in extracts prepared from the mouse skeletal muscle, spinal cord, and brain that co-migrated with the bacterially expressed SMN protein. In immunohistochemical studies, the anti-SMN antibody labeled mostly the cytoplasm of the motor neurons in the anterior horn of mouse spinal cord. In contrast, predominant uniform labeling of the nuclei was observed in the mouse skeletal muscle. Thus, our results for the first time demonstrate that the SMN protein is differentially localized in mouse spinal cord and skeletal muscle.

Oxidation of the skeletal muscle Ca2+ release channel alters calmodulin binding.

This study presents evidence for a close relationship between the oxidation state of the skeletal muscle Ca2+ release channel (RyR1) and its ability to bind calmodulin (CaM). CaM enhances the activity of RyR1 in low Ca2+ and inhibits its activity in high Ca2+. Oxidation, which activates the channel, blocks the binding of 125I-labeled CaM at both micromolar and nanomolar Ca2+ concentrations. Conversely, bound CaM slows oxidation-induced cross-linking between subunits of the RyR1 tetramer. Alkylation of hyperreactive sulfhydryls (<3% of the total sulfhydryls) on RyR1 with N-ethylmaleimide completely blocks oxidant-induced intersubunit cross-linking and inhibits Ca2+-free 125I-CaM but not Ca2+/125I-CaM binding. These studies suggest that 1) the sites on RyR1 for binding apocalmodulin have features distinct from those of the Ca2+/CaM site, 2) oxidation may alter the activity of RyR1 in part by altering its interaction with CaM, and 3) CaM may protect RyR1 from oxidative modifications during periods of oxidative stress.

Role of receptor and protein kinase C activation in the internalization of the gastrin-releasing peptide receptor.

The mechanisms regulating receptor internalization are not well understood and vary among different G protein-coupled receptors. The bombesin (Bn)/gastrin-releasing peptide receptor GRP-R, which is coupled to phospholipase C via the Gq family of transducing proteins, is internalized rapidly after Bn binding. Agonist stimulation leads to rapid receptor phosphorylation, as does activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA). However, agonist- and PMA-induced phosphorylation occur at different receptor sites. Here, we examined the role of PKC in GRP-R internalization after agonist and antagonist binding. We synthesized [D-Tyr6]Bn(6-13)propylamide ([D-Tyr6]Bn(6-13)PA) and found that it potently inhibited Bn-stimulated insulin release and [125I-Tyr4]Bn binding (Ki = 4.72 nM) in the HIT-T15 pancreatic cell line. The radiolabeled antagonist peptide, [125I-D-Tyr6]Bn(6-13)PA, bound with high affinity (KD = 0.29 nM at 4 degrees) to a single class of receptor sites, and competition binding studies exhibited the analog specificity expected for the GRP-R subtype. Although the agonist [125I-Tyr4]Bn was internalized rapidly at 37 degrees and subsequently degraded, [125I-D-Tyr6]Bn(6-13)PA was not internalized and was released into the medium mainly as intact peptide. The lysosomal inhibitor chloroquine (200 microM) increased the intracellular accumulation of [125I-Tyr4]Bn but had no effect on the subcellular distribution of [125I-D-Tyr6]Bn(6-13)PA. Consistent with these observations, the treatment of cells with 100 nM Bn at 37 degrees reduced cell surface receptors within minutes, whereas [D-Tyr6]Bn(6-13)PA had no effect. The addition of PMA did not induce the internalization of antagonist-occupied receptors, but pharmacological inhibition of PKC decreased the rate of agonist-induced receptor internalization. These results therefore demonstrate that although PKC contributes to agonist-induced internalization of the GRP-R, it does not elicit receptor internalization of the antagonist-occupied receptor.

Agonist binding and protein kinase C activation stimulate phosphorylation of the gastrin-releasing peptide receptor at distinct sites.

Gastrin-releasing peptide and other bombesin-like peptides stimulate secretion, cell proliferation, and smooth muscle contraction via a family of G protein-coupled receptors that activate phospholipase C. Second messenger formation by one of these receptors, called BR1, is rapidly desensitized after treatment of cells with either agonists or the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine whether receptor phosphorylation was involved in BR1 desensitization, we generated antibodies to a peptide corresponding to a unique sequence within the COOH terminus of this receptor. One antibody (BR1-517) immunoprecipitated 60% of the solubilized [125I-Tyr4]bombesin/receptor complex prepared from either Swiss 3T3 fibroblasts or CHO-K1 cells transfected to express high levels of mouse BR1 (CHO-mBR1). Furthermore, immunoprecipitation of photoaffinity-labeled receptors yielded the expected 87-kDa radiolabeled band on gel electrophoresis. Phosphorylation of this immunoprecipitated receptor protein was markedly stimulated when [32P]orthophosphate-labeled Swiss 3T3 cells or CHO-mBR1 cells were treated with 100 nM bombesin for 5 min. 32PO4 incorporation into immunoprecipitated receptor was detectable after 2 min and maximal after 15 min of bombesin treatment. Phosphoamino acid analysis showed 32P labeling of serine and theonine but not tyrosine residues. Pretreatment of CHO-mBR1 cells with 100 nM TPA for 30 min also desensitized bombesin stimulation of inositol-1,4,5-trisphosphate formation. However, TPA did not increase 32PO4 incorporation into the immunoprecipitated receptor, although protein kinase C inhibition potentiated bombesin-induced receptor phosphorylation. Subsequent studies showed that TPA did stimulate receptor phosphorylation, but the antibody did not recognize this phosphorylated state of the receptor. Thus, TPA decreased the efficiency of receptor immunoprecipitation, and subsequent incubation of receptor with alkaline phosphatase reversed this TPA inhibition. The differential specificity of the antibody for various phosphorylated forms of BR1 demonstrates that agonist-induced and TPA-induced phosphorylations of the receptor occur at distinct sites.

Bombesin receptors in a human duodenal tumor cell line: binding properties and function.

The bombesin family of peptides elicit numerous biological responses in the gut, including stimulation of cell proliferation, and have been implicated as growth factors in a variety of gastrointestinal tumors. Even though these peptides and their receptors are distributed throughout the gastrointestinal tract, there are few cell lines available as model systems to study bombesin action in gastrointestinal cells. In this study, we have characterized functional bombesin receptors in a human duodenal cancer cell line, HuTu-80. The binding of [125I-Tyr4]bombesin to intact cells at 4 degrees C reached equilibrium by 6 h. Scatchard analysis of [125I-Tyr4]bombesin binding showed that HuTu-80 cells contained a single class of high affinity binding sites (5900 +/- 1960/cell; Kd = 80 +/- 20 pM). [125I-Tyr4]bombesin binding was inhibited by bombesin receptor agonists and antagonists with the following order of potencies: gastrin-releasing peptide (GRP) = GRP-(14-27) = bombesin > [DPhe6]bombesin(6-13)ethylamide > [Leu13 psi-(CH2NH)Leu14]bombesin > neuromedin B. Photoaffinity cross-linking studies, in which N-5-azido-2-nitrobenzoyloxysuccinimide was used to covalently couple [125I]GRP(14-27) to cells at 4 degrees C, resulted in the specific labeling of a broad band with an apparent molecular mass of 66,000 daltons. Consistent with the presence of high affinity receptors, bombesin increased the formation of inositol phosphates in HuTu-80 cells in a dose-dependent manner (concentration eliciting half-maximal effect, 290 +/- 70 pM). However, under conditions where both insulin and serum increased [3H]thymidine incorporation into DNA, 10 nM bombesin had no effect either alone or in the presence of insulin. Bombesin also had no effect on colony formation by HuTu-80 cells in soft agar. Furthermore, the bombesin receptor antagonist, [Leu13 psi(CH2NH)Leu14]bombesin, did not inhibit [3H]thymidine incorporation or clonal growth either in the absence or in the presence of serum. Together, these results show that HuTu-80 cells contain high affinity bombesin receptors of the GRP subtype. These receptors are functionally coupled to second messenger production but do not stimulate cell proliferation.