Lucia Vallar

Mechanisms of signal transduction at the dopamine D2 receptor

D2 dopamine receptor activation induces inhibition of adenylate cyclase, with a rapid decrease of cAMP levels, and an ensuing blockade of IP3-dependent release of Ca2+ from intracellular stores. K+ channels are concomitantly activated and Ca2+ channels are possibly also inhibited. The increased K+ conductance causes hyperpolarization, which may be responsible for the abolition of Ca2+ action potentials and [Ca2+]i fluctuations occurring both at rest and after activation of receptors coupled to PIP2 hydrolysis. Lucia Vallar and Jacopo Meldolesi analyse this spectrum of intracellular signals which might be sufficient to sustain inhibition of secretion in pituitary lactotroph cells and possibly the other effects of D2 receptors in other cell systems.

G protein oncogenes in pituitary tumors

G proteins are involved in the transduction of external signals from cell surface receptors to intracellular effectors. Somatic mutations activating the a-subunit of G(s) (the stimulatory regulatory protein of adenylyl cyclase) by inhibiting its intrinsic GTPase activity have been first identified in human GH-secreting adenomas and subsequently found in thyroid tumors and in McCune-Albright syndrome. It has been therefore proposed that the gene encoding the GS a-subunit may be converted into an oncogene (gsp for GS protein) in cell types that proliferate in response to cAMP. Since several G proteins mediate signaling pathways that are effective in coupling external stimuli to cell proliferation, it appears most likely that in the near future other G protein oncogenes will be identified in human tumors.

Regulation of exocytosis in electrically permeabilized insulin-secreting cells. Evidence for Ca2+ dependent and independent secretion

The regulation of insulin secretion from RINm5F cells exposed to high voltage discharge has been investigated. Electron microscopy revealed that the overall structure of the cells was preserved after permeabilization. In this preparation insulin release was stimulated by Ca2+ (EC50=2.4 μM). The stable GTP analogue GTPγS enhanced secretion both at intermediate (nano- to micromolar) and vanishingly low (<10 pM) Ca2+ concentrations. At optimal Ca2+ (10 μM) the effect of GTPγS was greatly reduced. We investigated whether the secretory response to GTP analogues was mediated by any of three enzyme systems regulated by GTP-binding proteins, i.e. generation of cyclic AMP by adenylate cyclase, of diacylglycerol by phospholipase C and of arachidonic acid by phospholipase A2. The involvement of these messenger systems could be excluded as (i) cyclic AMP only had minor, Ca2+ dependent effects, (ii) phospholipase C was not activated in the absence of Ca2+ and insulin secretion due to the phorbol ester TPA displayed a different Ca2+ dependency, (iii) arachidonic acid did not elicit Ca2+ independent insulin secretion. These results, taken together with the finding that insulin secretion due to Ca2+ or TPA is attenuated by the inhibitory guanine nucleotide GDPβS, suggest the existence of a regulatory site in exocytosis which is sensitive to guanine nucleotides.

G-Protein Oncogenes in Acromegaly

G-proteins belong to a family of proteins which share the common properties of GTP binding and hydrolysis. Heterotrimeric G-proteins are composed of alpha-, beta- and gamma-subunits. The alpha-subunit which differs from one G-protein to another contains the GDP/GTP binding site and has intrinsic GTPase activity. The receptor occupancy causes displacement of bound GDP by GTP, dissociation of free beta gamma-dimer and alpha-GTP complex, interaction of the activated alpha-GTP complex with intracellular effectors, such as enzymes and ion channels. The turn off of the reaction is due to the GTPase activity which causes the hydrolysis of GTP to GDP. G-proteins are essential for transferring hormonal signals from cell surface receptors to intracellular effectors. Since G-proteins generate intracellular effectors involved in cell growth, G-protein genes have the propensity to be converted into oncogenes. In fact, mutations in the alpha-subunit of Gs (the G-protein involved in the activation of adenylyl cyclase) have been demonstrated in 40% of human GH secreting pituitary adenomas. Single amino acid substitutions replacing Arg 201 with either Cys or His or Gln 227 with either Arg or Leu cause constitutive activation of adenylyl cyclase by inhibiting GTPase (gsp oncogene). The same mutations were identified in about 10% of thyroid adenomas and in the McCune-Albright syndrome.

In vitro studies on TSH secretion and adenylate cyclase activity in a human TSH-secreting pituitary adenoma. Effects of somatostatin and dopamine

We have studied the in vitro TSH secretion and the adenylate cyclase (AC) activity of a human pituitary adenoma surgically removed from a hyperthyroid patient showing high serum TSH levels. The tumor appeared almost homogeneously constituted by cells positive for an anti-TSH-beta antiserum and showing the ultrastructural characteristics of the adenomatous thyrotrophs. Adenoma fragments released in vitro a large amount of TSH (148.4 μU/mg prot/30 min), alpha-subunit(35.5 ng/mg prot/30 min) and TSH-beta (10.1 ng/mg prot/30 min). The effects of somatostatin (GHRIH) and dopamine (DA) on the hormone release have been tested in vitro. Both agents markedly inhibited the release of intact TSH and TSH-beta whereas the release of alpha-subunit was less affected. The two agents were effective at concentrations higher than 10−8M. The ability of GHRIH and DA in modulating the AC activity was investigated in membrane fraction preparations. GHRIH inhibited AC at concentrations higher than 10 −7M. The maximal inhibition was 32% at 10−5 M. Conversely, DA slightly stimulated AC activity. This effects was not mimicked by the dopaminergic ergot CH 29–717, which was completely ineffective on the enzyme. These results suggest that: 1) in this TSH-secreting pituitary adenoma a normal secretory response to the inhibiting agents (GHRIH and DA) is present; 2) different mechanisms of transduction of the GHRIH and DA signals (cAMP dependent and cAMP independent) could be operating in this tumor.

G-Proteins and Hormonal Signalling in Human Pituitary Tumors: Genetic Mutations and Functional Alterations

In the last few years, molecular studies on pituitary adenomas have yielded several lines of evidence supporting a primary pituitary origin for these tumors. In fact, analyses of x-chromosomal inactivation show that the great majority of pituitary tumors are monoclonal in origin, suggesting that one or more mutations are responsible for the selective expansion of a single cell clone. Mutations constitutively activating GTP-binding proteins have been identified in subsets of pituitary adenomas. Single amino acid substitutions replacing Arg 201 with either Cys, His, or Gln 227 with either Arg or Leu of the alpha-subunit of the Gs gene were identified in one third of growth hormone (GH)-secreting adenomas. Both mutations stabilize alpha s in its active conformation by inhibiting GTPase activity, thus mimicking the effect of specific extracellular growth factors, such as growth hormone releasing hormone (GHRH). Since several lines of evidence suggest that cAMP is involved in somatotrope replication, it has been proposed that the alpha s gene can be converted into an oncogene, designated gsp (for Gs protein). Recently, the ras oncogene has been identified in one prolactinoma characterized by unusual invasiveness. Although these data seem to negate a primary role for hypothalamic neurohormones in adenoma formation, it is conceivable that the hormones may exert a role in the sequence of events leading to clonal expansion of a transformed cell. Moreover, alterations in receptor and/or postreceptor events triggered by hypothalamic neurohormones may result in amplification of stimulatory inputs and impairment of inhibitory ones.

Protein homeostasis in neurons and its pathological alterations.

In neuronal cells, proteins are synthesized on ribosomes from the genetic information encoded in DNA. In some instances translation takes place at the neuronal cell soma but in other it occurs at distal location, such as in a dendritic spine. Folding is usually initiated before the completion of protein synthesis and its outcome strictly depends on the local environment in which the nascent protein is submerged. Incompletely folded proteins and, more importantly, misfolded proteins are under the surveillance of several quality control systems that re-establish the correct conformation or initiate protein degradation. Regulation and maintenance of these systems is a vital issue for neuronal and glial cells, and impairments at different levels leads to neurodegenerative diseases.

Mutations in the Alpha Subunit of the Stimulatory Regulatory Protein of Adenylyl Cyclase (Gs) in Human GH-Secreting Pituitary Adenomas: Biochemical, Clinical, and Morphological Aspects

Very recently a subset of human GH-secreting pituitary adenomas carrying a somatic mutation in the alpha subunit of the stimulatory regulatory protein of adenylyl cyclase (Gs) was identified. In all these tumors (Group 2; about 30% of all the GH secreting tumors studied) the alpha s cDNAs contained mutations; in 8 tumors mutations replaced Arginine 201 with either Cystein or Histidine while in the remaining tumors Glutamine 227 was replaced by either Arginine or Leucine. No mutations were observed in the remaining adenomas (Group 1). The two mutations caused a constitutive activation of adenylyl cyclase and a turn on of cAMP synthesis by inhibiting GTPase activity. The transformed phenotype was reflected in adenomatous cells with high rate of cAMP production and in vitro GH secretion. No difference in age, sex, clinical features, duration of the disease and cure rate were observed between the patients without (Group 1) or with alpha s mutation (Group 2), while higher serum GH levels and smaller tumor size were present in Group 2 patients. Moreover, hypersecretory activity in Group 2 tumors was also apparent at electron microscopy; cells of Group 2 tumors were densely granulated and showed prominent rough endoplasmic reticulum and Golgi complex. With respect to Group 1, Group 2 patients were less responsive to GH-releasing hormone (GHRH), while they were more sensitive to somastostatin. The former finding is in agreement with the hypothesis that the oncogenic proteins mimic the effects of extracellular growth factors, so removing the requirement for GHRH; the latter might explain the low rate of tumor growth as due to the counteracting role of endogenous inhibitory factors.

The hypersecreting somatotroph: functional and morphological aspects

Patients with acromegaly often show peculiar alterations of the GH secretory pattern. In particular, alterations in the sensitivity to physiological releasing (GHRH) or inhibiting (SRJF) hormones, together with abnormal responsiveness to hypothalamic agents such as dopamine (DA) and TRH or GnRH, are frequently observed in this disease. Cosecretion of pituitary hormones other than GH, such as prolactin, glycoprotein α-subunit, and TSH, may also occur. However, the underlying biochemical events and morphological correlates have not so far been extensively investigated. This prompted us to investigate the following aspects: — Alterations of receptor regulation — Alterations of postreceptor “coupling” processes — Transmembrane signaling pathology — Colocalization of different hormones in the same cell

Independent Regulation of Rap1 and Mitogen-Activated Protein Kinase by the α Chain of Go

Receptors coupled to Gi/o proteins stimulate the mitogen-activated protein kinase (MAPK) cascade. The intracellular pathways linking the α chains of these G proteins to MAPK activation are not completely understood. One of the signaling molecules which has been suggested to act downstream of Gαi/o is the small G protein Rap1. We investigated the role of Rap1 in MAPK stimulation by Gαo in Chinese hamster ovary (CHO) cells. Our previous results have shown that in this cell system activated Gαo strongly potentiates the MAPK response to the epidermal growth factor (EGF) receptor. Rap1 regulation was examined in cells transfected with Rap1 and wild-type Gαo or the activated mutant Gαo-Q205L. Immunocytochemical analysis detected both Rap1 and the Gαo subunit at the plasma membrane as well as on perinuclear cytoplasmic vesicles. Expression of wild-type Gαo had no significant effect on the levels of activated Rap1. In contrast, Gαo-Q205L virtually abolished the activation of Rap1 induced by EGF. Further experiments showed that MAPK stimulation by EGF was greatly inhibited by expression of activated Rap1, suggesting that Rap1 inhibition could mediate the effect of Gαo on the MAPK cascade. However, Gαo-Q205L efficiently inhibited the activation of Rap1 induced by fibroblast growth factor (FGF). We have previously found that the ability of FGF to activate MAPK is not modified by Gαo. In addition, expression of the GAP protein RAP1GAPII blocked Rap1 activation without affecting EGF- or FGF-dependent MAPK stimulation. These findings provide evidence for independent regulation of Rap1 and MAPK by the Go α chain.