Investigators in the Endocrinology and Reproductive Research Branch address the molecular and cellular mechanisms of reproductive hormone action, the structure-function properties of hormone receptors, and the mechanisms and roles of protein phosphorylation and sulfonation in metabolic regulation and signal transduction. The branch found that the manner in which the endogenous GnRH receptor mediates regulation of pulsatile neuropeptide secretion involves autocrine switching of receptor coupling between stimulatory and inhibitory G proteins. The LH receptor gene promoter is under tonic repression by deacetylation and is activated by depression through specific signal inputs. The HDAC-mSin3A complex regulates human LHR gene transcription by interacting with Sp1/Sp3 and by region-specific changes in histone acetylation and Pol II recruitment to the promoter. The complex genomic structure of the human Prl receptor gene has been resolved, and the manner in which its transcription is controlled by multiple promoters with individual regulatory mechanisms is undergoing clarification. Identification of the enzyme that sulfonates cholesterol revealed two isoforms derived by differential splicing that preferentially produce cholesterol sulfate or pregnenolone sulfate.

Investigations of calcium signalling in pituitary cells explored the role of endothelin receptors in controlling voltage-gated calcium influx, the extracellular messenger functions of calcium, and the interactions between intracellular calcium and protein kinase A in the control of soluble guanylyl cyclase activity. Studies on calcium-conducting purinergic receptors have identified structural domains that determine ligand selectivity, desensitization, and recovery from desensitization. Heteromeric receptors formed from distinct subunits were found to exhibit new functions that are endowed by the participating extracellular and C-terminal subdomains.

The branch has advanced the analysis of molecular mechanisms of cell signalling through the use of fluorophor-labeled proteins. These probes permit the visualization of highly localized changes in inositol lipid levels in cellular responses ranging from ovum fertilization to phagocytosis of bacteria, and has enabled the group to visualize the active form of the oncogenic protein, Ras, and to identify the vesicular compartments through which the agonist-activated angiotensin AT1 receptor is internalized and recycled to the plasma membrane.

The branch investigates the physical and functional interactions between G protein–coupled receptors (GPCRs) and growth factor receptors in neuronal, pituitary, and hepatic cells. Analysis of the complex pathways that mediate the activation of MAP kinase phosphorylation cascades has identified temporal signalling modalities that are determined by transactivation of the EGF receptor by GPCRs such as the angiotensin and GnRH receptors. These studies are directed at understanding the molecular mechanisms that determine the intensity and spatio-temporal regulation of ERK signalling and its actions on nuclear gene expression during mitogenic signalling by GPCRs.

The importance of neurogranin, a calmodulin (CaM) binding protein, in synaptic plasticity has been shown in knock-out mice with impaired learning as well as deficits in long-term potentiation and CaM kinase autophosphorylation. Both phosphorylation and oxidation of neurogranin regulate neuronal signalling during NMDA receptor activation. Thionylation of neuronal proteins provides protection against oxidative insults and acts in cell signalling. Glutathione disufide S-oxide has been identified as a highly reactive glutathionylating agent and can serve to integrate oxidative and nitrosative cellular responses through its reaction with thiol groups.