A capability for scanning electron microscopy of wet biological specimens is presented. A membrane that is transparent to electrons protects the fully hydrated sample from the vacuum. The result is a hybrid technique combining the ease of use and ability to see into cells of optical microscopy with the higher resolution of electron microscopy. The resolution of low-contrast materials is Ϸ100 nm, whereas in high-contrast materials the resolution can reach 10 nm. Standard immunogold techniques and heavy-metal stains can be applied and viewed in the fluid to improve the contrast. Images present a striking combination of whole-cell morphology with a wealth of internal details. A possibility for direct inspection of tissue slices transpires, imaging only the external layer of cells. Simultaneous imaging with photons excited by the electrons incorporates data on material distribution, indicating a potential for multilabeling and specific scintillating markers.

Analogs of parathyroid hormone (PTH)- The parathyroid hormone (PTH)1 /PTH-related protein (PTHrP) receptor (PTH1R) is a G protein-coupled, seven-transmembrane domain-containing receptor (GPCR) activated equipotently by two distinct hormones, PTH and PTHrP (1, 2). Although acting through the same receptor, these hormones have different physiological functions: PTH is a regulator of blood calcium levels (3), whereas PTHrP is an autocrine/paracrine factor involved in skeletal and cartilage development (4, 5). The PTH1R is a member of the glucagon/secretin/calcitonin subfamily of GPCRs (2). It is coupled to both adenylyl cyclase (AC)/cyclic AMP and phospholipase C/inositol 1,4,5-trisphosphate/cytosolic calcium intracellular signaling pathways (6 -8).Extensive efforts have been focused on investigating the structural basis for hormone recognition by PTH1R. These lines of research have identified regions in both the ligands and the receptor required for binding and signaling. On one hand, the mutagenesis approach, generating chimeric, mutated, or truncated receptors, has established that multiple receptor domains are involved in the complex interaction with the ligands, including the N-terminal extracellular tail, extracellular loops, and transmembrane domains (TMDs) (9 -11). On the other hand, structure-function studies of the hormones and their analogs have revealed structural determinants required for interaction with the receptor. These studies have identified the C-terminal region of PTH-(1-34) and PTHrP-(1-34) as the principal binding domain (12-14), which is also required for activation of protein kinase C (15, 16). The N-terminal 1-6 sequence of either ligand was found to function as the principal activation domain (Ref. 17 and references therein).The complementary biochemical approach of photoaffinity cross-linking has been utilized to examine directly ligand-receptor bimolecular interactions through the generation of covalently linked radiolabeled ligand-receptor photoconjugates. Here, we report the evaluation of a series of photoreactive analogs of PTHrP in order to probe the nature of receptor interaction with the principal activation domain of the hormone (residues 1-6).

Endocytosis and intracellular trafficking of the human parathyroid hormone receptor subtype 1 (hPTH1-Rc) and its ligands was monitored independently by real-time fluorescence microscopy in stably transfected HEK-293 cells. Complexes of fluorescence-labeled parathyroid hormone (PTH)-(1-34) agonist bound to the hPTH1-Rc internalized rapidly at 37°C via clathrincoated vesicles, whereas fluorescent PTH-(7-34) antagonist-hPTH1Rc complexes did not. A functional C terminus epitope-tagged receptor (C-Tag-hPTH1-Rc) was immunolocalized to the cell membrane and, to a lesser extent, the cytoplasm. PTH and PTH-related protein agonists stimulated C-Tag-hPTH1-Rc internalization. Relocalization to the cell membrane occurred 1 h after removal of the ligand. Endocytosis of fluorescent PTH agonist-hPTH1-Rc complexes was blocked by the protein kinase C (PKC) inhibitor staurosporine but not by the specific protein kinase A inhibitor N-(2-(methylamino)ethyl)-5-isoquinoline-sulfonamide. Fluorescent PTH antagonist-hPTH1-Rc complexes were rapidly internalized after PKC activation by phorbol 12-myristate 13-acetate or thrombin, but not after stimulation of the cAMP/protein kinase A pathway by forskolin. In cells co-expressing the hPTH1-Rc and a green fluorescent protein-␤-arrestin2 fusion protein (␤-Arr2-GFP), PTH agonists stimulated ␤-Arr2-GFP mobilization to the cell membrane. Subsequently, fluorescent PTH-(1-34)-hPTH1Rc complexes and ␤-Arr2-GFP co-localized intracellularly. In conclusion, agonist-activated hPTH1-Rc internalization involves ␤-arrestin mobilization and targeting to clathrin-coated vesicles. Our results also indicate that receptor occupancy, rather than receptor-mediated signaling, is necessary, although not sufficient, for endocytosis of the hPTH1-Rc. Activation of PKC, however, is absolutely required. G protein-coupled receptors (GPCRs)1 represent a major class of membrane-bound proteins that mediate a wide variety of biological functions, including expression of the biological actions of various hormones. The responsiveness of GPCRs to extracellular stimuli, and particularly to their natural ligands, is regulated by several mechanisms, including receptor phosphorylation, coupling and uncoupling from G proteins, receptor internalization (endocytosis), and regulation of receptor gene transcription (1-3). In particular, a model for agonist-activated GPCR endocytosis, which involves mobilization of ␤-arrestins followed by agonist-receptor complex internalization through clathrin-coated pits, has been well characterized for the ␤2-adrenergic receptor (4 -6). Some of these key features are common to other GPCRs (7-9), but notable exceptions exist. For example, although the angiotensin receptor AT1 is rapidly internalized in response to agonist stimulation, the homologous AT2 receptor is not (10). Although the -opioid receptor agonist etorphin promotes ␤-arrestin mobilization and receptor internalization, morphin does not (11). Moreover, in contrast to the widely observed requirement for receptor occupancy to enable endocyto...