Substance P (SP) is an undecapeptide whose functions are as varied as its locations. In the nervous system, it is thought to act as a neurotransmitter. In the peripheral vasculature, it has hypotensive effects and it causes contraction in the smooth muscle of the gut. In salivary gland, it is a potent secretagogue and it is how this effect is transduced that is the subject of this dissertation. Activation of the SP receptor in rat submaxillary gland by SP results in the hydrolysis of inositol phospholipids and the mobilization of intracellular Ca2+. These second messengers are then able to activate a pathway(s) which results in the secretion of electrolytes, water and macromolecules. The production of these second messengers, however, is thought to require the participation of a guanine nucleotide binding protein (G protein). The G protein that couples to the SP receptor (Gp), has not yet been identified. Although several investigators have recently reported the purification of G protein α subunits that are capable of activating phospholipase C, it is not known if they couple to receptors in order to activate phospholipase C. In an effort to learn more about the mechanisms of signal transduction by SP in salivary gland, the interactions of the SP receptor with G proteins were studied. In the first study, the question of whether the SP receptor functionally couples to a G protein was investigated. Alkaline treatment was used to deplete membranes containing SP receptors of endogenous G proteins. These membranes were not capable of binding SP with high affinity. High affinity binding capability was restored in those membranes, however, by reconstituting them with exogenous G proteins. Thus, it was concluded that that SP receptor agonist affinity is regulated by a G protein. It was also determined that the G proteins (a Go/Gi mixture) used to reconstitute the membranes may not be those that couple to the SP receptor in vivo, since the reconstituted Go/Gi mixture was inactivated by treatment with pertussis toxin, while Gp was not. The next study was undertaken in an effort to identify other G proteins that are able to interact with the SP receptor. G proteins were chromatographically purified from horse submaxillary gland membranes, and assayed for characteristics that could identify one or more G proteins as potential physiological couplers to the SP receptor. G proteins were identified in fractions by the ability to bind [35S]GTPγS. These GTP-binding proteins were further characterized by testing their susceptibility to ADP- ribosylation catalyzed by pertussis toxin and their ability to restore high affinity agonist binding in membranes containing the SP receptor, but no endogenous G proteins. In addition to identifying G proteins that are substrates for pertussis toxin-catalyzed ADP-ribosylation (e.g. Go and/or Gi), a GTP-binding protein was identified which possesses characteristics that are unlike those of the well-known G proteins, Go, Gi and Gs. This protein elutes from anion exchange resins at a high salt concentration, is not susceptible to ADP- ribosylation catalyzed by pertussis toxin, is able to reconstitute high affinity binding in G protein depleted rat submaxillary gland membranes and is not recognized by antibodies to Go, Gi, Gs or Gz. Finally, a direct characterization of the G protein coupled to the SP receptor in rat submaxillary gland was undertaken. Using photo-affinity labelling techniques in conjunction with chemical crosslinking techniques, a covalent 96 kDa SP receptor complex was identified. The generation of this 96 kDa complex was inhibited by a nonhydrolyzable analog of GTP, but not a nonhydrolyzable analog of ATP. Furthermore, the complex could not be produced in membranes that had been depleted of G proteins by alkaline treatment. Reversal of the chemical crosslink yielded only the 53 kDa SP receptor, showing that the protein crosslinking to the SP receptor possesses a molecular weight of about 43 kDa. This molecular weight is typical of G protein α subunits. It was concluded that the 96 kDa crosslinked receptor complex consisted of the SP receptor, the radioiodinated SP derivative and the α subunit of Gp. The studies show that the SP receptor may be coupled to a novel G protein, whose purification characteristics differ from those of the known G proteins. Although Gp has yet to be identified, comparisons of the results of these investigations with those of several recent articles in which the purification of G protein α subunits that are capable of stimulating phospholipase C is reported, suggests that Gp is similar, if not identical to those proteins. Furthermore, this dissertation describes a unique reconstitution system and crosslinking techniques which should prove useful in the identification of Gp, as well as in the study of other receptor-G protein interactions and perhaps, the reconstitution of the receptor-G protein-phospholipase C signal transduction pathway.

Type II 5'-deiodinase (D2) catalyzes the conversion of T4 to the transcriptionally active T3. When T4 levels are high, D2 activity levels are low. Conversely when T4 levels are low, D2 catalytic activity is high. Immunocytochemistry and biochemical data from cultured rat astrocytes revealed that physiological concentration of T4 and the non-transcriptionally active metabolite rT3, but not T3, initiates the budding of D2 containing endosomes and their subsequent translocation to the perinuclear space. Further analysis showed that this process required a polymerized actin cytoskeleton but not cellular transcription or translation; however the precise mechanism remained unknown. In this present investigation, we characterized the requirement of an unconventional myosin motor protein, myosin 5a, in the actin-based endocytosis of D2 containing vesicles. We developed an in vitro actin binding assay that exploited the T4 dependent binding of D2 containing vesicles to F-actin, and showed that D2p29:F-actin interactions are calcium, magnesium and ATP-dependent suggesting that a calmodulin (CaM) regulated myosin ATPase is required. Introduction of in vitro transcribed and translated vesicle-binding tail, which lacked the actin binding head, of myosin 5a to the in vitro actin binding assay created a dominant negative inhibitor of D2 binding to the actin cytoskeleton by competing with the native myosin 5a. A replication deficient adenoviral vector expressing the fusion protein of the 29 kDa substrate binding subunit of D2 with a green fluorescent protein reporter molecule enabled us to directly examine T4 dependent regulation of D2 in vitro as well as in living cells. Using immunoprecipitation we showed a T4 dependent association between the vesicle binding tail of myosin 5a and D2 containing vesicles. Biochemical analysis of the interaction of the myosin 5a tail with D2 containing vesicles revealed that the last 21 amino acids of myosin 5a were both necessary and sufficient for the attachment of D2 containing vesicles to the F-actin cytoskeleton. Using rapid acquisition time-lapse digital microscopy in p29GFP expressing rat astrocytes, we showed directed T4 dependent p29GFP movement from the plasma membrane to the perinuclear region. This hormone dependent vesicle movement was not observed in cells treated with T3 or no hormone. Time lapse motion studies allowed for the calculation of the velocity and of the distance traveled for individual fusion protein containing vesicles. The velocity for cells treated with T4 or rT3 was identical to that reported for vesicle-laden myosin 5a in mouse melanophores. In contrast cells treated with T3 or those receiving no hormone treatment had velocities similar to diffusion of proteins within the plasma membrane. Astrocytes constitutively expressing both p29GFP and dominant negative myosin 5a inhibitors failed to show hormone induced centripetal movement. These data demonstrate that myosin 5a is the molecular motor responsible for thyroid hormone dependent actin based endocytosis in astrocytes.