The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module

Abstract

Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of ϳ70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand-and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain.In transmembrane receptors a series of conformational changes are required to transmit the information of ligand binding (an extracellular signal) to the interior of the cell, resulting in either altered interaction with signaling intermediates or in the regulation of a catalytic activity present in the receptor. In these multidomain receptors, where the ligand binding and effector domains are present in the same polypeptide chain, the relay of conformational changes is under the exquisite control of post-translational modifications or precise structural alterations.Receptor guanylyl cyclases (GCs) 4 have an N-terminal extracellular ligand binding domain, a single transmembrane domain, and a C-terminal intracellular domain (1). Binding of ligands to the extracellular domain elicits a conformational change that increases the guanylyl cyclase activity of the receptor, resulting in increased cGMP production. The intracellular domain of receptor GCs contains a region that shares considerable sequence similarity to protein kinases and is referred to as the kinase homology domain (KHD). Binding of ATP to the KHD induces a conformational change that regulates cGMP production by the guanylyl cyclase domain (2). Thus, receptor GCs exemplify the intricate interactions between domains in transducing the signal from an extracellular ligand to the interior of the cell.The amino acid sequences of ...

Site-specific N-Linked Glycosylation of Receptor Guanylyl Cyclase C Regulates Ligand Binding, Ligand-mediated Activation and Interaction with Vesicular Integral Membrane Protein 36, VIP36

“…A single transmembrane domain is followed by a pseudokinase domain with sequence similarity to protein kinases (16,17). The C-terminal guanylyl cyclase domain is linked to the kinase homology domain by a linker region (18). We have shown that the kinase homology domain binds ATP and mutation of a critical lysine residue involved in interacting with the ␤-phosphate of ATP prevents ligand-mediated activation of GC-C (16).…”

“…We have shown that the kinase homology domain binds ATP and mutation of a critical lysine residue involved in interacting with the ␤-phosphate of ATP prevents ligand-mediated activation of GC-C (16). The linker region appears to adopt a helical structure and extensive mutational analysis demonstrated the critical requirement of the linker in repressing the guanylyl cyclase activity of the receptor in the absence of its ligands (18).…”

“…The concentrations of free metals and metal-GTP complexes in assays were calculated using MaxChelator.When MnGTP was used as a substrate, activity measured represents the full catalytic potential of the guanylyl cyclase domain in GC-C (18). When MgGTP was used as a substrate, the basal guanylyl cyclase activity was lower, and therefore, ligand-mediated activation could be readily detected (18). ST (10 Ϫ7 M) was added to measure ligand-mediated cGMP production, with MgGTP as substrate.…”

“…Heterologous Expression of Wild Type and Mutant GC-CWild type and mutant GC-C were expressed in HEK293T cells and membrane protein fractions were prepared as described previously (18). In some cases, monolayers of cells were treated with 20 g/ml of tunicamycin (Sigma-Aldrich) 18-h post transfection, and membrane fractions were prepared from these cells 24-h post treatment.…”

Receptor Guanylyl Cyclases in Sensory Processing

“…Systematic mutational and biochemical analyses of GC-C and GC-A have suggested an important role for the linker region in repressing the catalytic activity of the receptors in the absence of their ligands (133). Specific residues in the linker region seem to assist in repressing GC activity through its interaction with the GCD, where Gα may also interact with and activate (or inhibit) rGC.…”