Ca2+ differently affects hydrophobic properties of guanylyl cyclase-activating proteins (GCAPs) and recoverin.

Abstract

Guanylyl cyclase-activating proteins (GCAPs) and recoverin are retina-specific Ca(2+)-binding proteins involved in phototransduction. We provide here evidence that in spite of structural similarities GCAPs and recoverin differently change their overall hydrophobic properties in response to Ca(2+). Using native bovine GCAP1, GCAP2 and recoverin we show that: i) the Ca(2+)-dependent binding of recoverin to Phenyl-Sepharose is distinct from such interactions of GCAPs; ii) fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) is markedly higher at high [Ca(2+)](free) (10 microM) than at low [Ca(2+)](free) (10 nM) in the presence of recoverin, while an opposing effect is observed in the presence of GCAPs; iii) fluorescence resonance energy transfer from tryptophane residues to ANS is more efficient at high [Ca(2+)](free) in recoverin and at low [Ca(2+)](free) in GCAP2. Such different changes of hydrophobicity evoked by Ca(2+) appear to be the precondition for possible mechanisms by which GCAPs and recoverin control the activities of their target enzymes.

A Novel Approach to Bacterial Expression and Purification of Myristoylated Forms of Neuronal Calcium Sensor Proteins

“…Consistently, these proteins do not change or even decrease their surface hydrophobicity upon calcium binding [29,85] and Ca 2+ /affinity chromatography on Phenyl Sepharose is not applicable for their purification. Nevertheless, both GCAPs are generally very hydrophobic [29,85], and therefore, can be purified using Ca 2+ -independent hydrophobic interaction chromatography. This approach was employed in previous studies for GCAP1 purification: after the gel filtration step the protein was purified on Butyl Sepharose column without using salt gradient [86].…”

“…In contrast to recoverin and NCALD, GCAPs do not possess a functional Ca 2+ -myristoyl switch, and their myristoyl group is sequestered inside the protein globule, which makes the structure of their Ca 2+ -free and Ca 2+ -bound conformers less different (at least in case of GCAP1) [50,79]. Consistently, these proteins do not change or even decrease their surface hydrophobicity upon calcium binding [29,85] and Ca 2+ /affinity chromatography on Phenyl Sepharose is not applicable for their purification. Nevertheless, both GCAPs are generally very hydrophobic [29,85], and therefore, can be purified using Ca 2+ -independent hydrophobic interaction chromatography.…”

Photoreceptor guanylate cyclase variants: cGMP production under control.

“…In the mammalian retina, three GCAPs (GCAP1, GCAP2 and GCAP3) have been identified Palczewski et al, 1994;Dizhoor et al, 1995;Haeseleer et al, 1999;Kobialka & Gorczyca, 2000;Imanishi et al, 2002;Gorczyca et al, 2003) that regulate the activity of photoreceptor GCs in Ca 2+ -dependent manners. GCAPs belong to the family of recoverin-like proteins with limited homology to CaM, and they are myristoylated at the N-terminus .…”

“…GCs come in two varieties: soluble and membrane-bound GCs with multiple isoenzymes of both forms being expressed ubiquitously (Drewett & Garbers, 1994;Kobialka & Gorczyca, 2000;Gorczyca et al, 2003). The membrane-bound GCs display similar topologies and belong to a family of single transmembrane-spanning signaling receptors (Singh et al, 1988) (Fig.…”

Metabolism of cyclic GMP in peritoneal macrophages of rat and guinea pig.

“…Three isoforms (GC-A, GC-B, and GC-C) of particulate cyclases are receptors for peptide hormones and are activated either by natriuretic peptides (GC-A, GC-B) or by guanylins and bacterial heat-stable enterotoxins (GC-C) (Kobia³ka & Gorczyca, 2000;Vaandrager, 2002). Two other particulate cyclases (GC-E and GC-F), found mainly in the photoreceptor cells of the vertebrate retina, are regulated by intracellular interaction with calcium-binding proteins named guanylyl cyclase-activating proteins (Dejda et al, 2002;Gorczyca et al, 2003;Kobia³ka & Gorczyca, 2000). Cyclic nucleotide phosphodiesterases catalyze hydrolysis of cyclic 3¢,5¢-nucleoside monophosphates (cAMP and/or cGMP) to respective 5¢-nucleoside monophosphates.…”