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Abstract
Membrane guanylate cyclase (MGC) is a ubiquitous multi-switching cyclic GMP generating signaling machine linked with countless physiological processes. In mammals it is encoded by seven distinct homologous genes. It is a single transmembrane spanning multi-modular protein; composed of integrated blocks and existing in homo-dimeric form. Its core catalytic domain (CCD) module is a common transduction center where all incoming signals are translated into the production of cyclic GMP, a cellular signal second messenger. Crystal structure of the MGC’s CCD does not exist and its precise identity is ill-defined. Here, we define it at a sub-molecular level for the phototransduction-linked MGC, the rod outer segment guanylate cyclase type 1, ROS-GC1. (1) The CCD is a conserved 145-residue structural unit, represented by the segment V820-P964. (2) It exists as a homo-dimer and contains seven conserved catalytic elements (CEs) wedged into seven conserved motifs. (3) It also contains a conserved 21-residue neurocalcin δ-modulated structural domain, V836-L857. (4) Site-directed mutagenesis documents that each of the seven CEs governs the cyclase’s catalytic activity. (5) In contrast to the soluble and the bacterium MGC which use Mn2+-GTP substrate for catalysis, MGC CCD uses the natural Mg2+-GTP substrate. (6) Strikingly, the MGC CCD requires anchoring by the Transmembrane Domain (TMD) to exhibit its major (∼92%) catalytic activity; in isolated form the activity is only marginal. This feature is not linked with any unique sequence of the TMD; there is minimal conservation in TMD. Finally, (7) the seven CEs control each of four phototransduction pathways- -two Ca2+-sensor GCAPs-, one Ca2+-sensor, S100B-, and one bicarbonate-modulated. The findings disclose that the CCD of ROS-GC1 has built-in regulatory elements that control its signal translational activity. Due to conservation of these regulatory elements, it is proposed that these elements also control the physiological activity of other members of MGC family.
Highlights
At the time of discovery (Paul et al, 1987) and molecular cloning of the first membrane guanylate cyclase (MGC), ANFRGC’s (Chinkers et al, 1989; Lowe et al, 1989; Duda et al, 1991; reviewed in Sharma, 2002, 2010) hydropathic analysis predicted that the protein is composed of three general domains: Extracellular (ExtD), Transmembrane (TMD), and Intracellular (ICD)
It was later determined that the region beyond Y965 of ROS-GC1 does not contribute to the cyclase catalytic activity (Duda et al, 2002), this residue marks the catalytic domain (CCD) C-terminal boundary
To verify the precision of setting the ROS-GC1 CCD boundaries to G817 and Y965 as its N- and C-termini, its sequence was aligned with the corresponding CCDs of Cyg12 and Cya2, the first crystalized guanylate cyclase catalytic domains
Summary
At the time of discovery (Paul et al, 1987) and molecular cloning of the first membrane guanylate cyclase (MGC), ANFRGC’s (Chinkers et al, 1989; Lowe et al, 1989; Duda et al, 1991; reviewed in Sharma, 2002, 2010) hydropathic analysis predicted that the protein is composed of three general domains: Extracellular (ExtD), Transmembrane (TMD), and Intracellular (ICD). It was proposed that this inter-domain region, wedged between the KHD and the catalytic domain, constitutes the dimerization domain (DD) (Garbers, 1992). It is conserved among the MGC family and functionally causes dimerization of the catalytic domain transforming it into catalytically active form (Wilson and Chinkers, 1995). This concept was broadened and applied to define the mechanism by which it regulates the activity of ROS-GC1 (Ramamurthy et al, 2001). The central theme of this concept was that the native isolated catalytic domain exists in its inactive form and DD transforms it into an active dimeric form
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