Sst 2A Research Articles

Pasireotide and Octreotide Stimulate Distinct Patterns of sst2A Somatostatin Receptor Phosphorylation

Pasireotide (SOM230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, Cushing's disease, and carcinoid tumors. Whereas octreotide acts primarily via the sst(2A) somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst(2A) activity combined with enhanced binding to other somatostatin receptor subtypes. In the present study, we used phophosite-specific antibodies to examine agonist-induced phosphorylation of the rat sst(2A) receptor. We show that somatostatin and octreotide stimulate the complete phosphorylation of a cluster of four threonine residues within the cytoplasmic (353)TTETQRT(359) motif in a variety of cultured cell lines in vitro as well as in intact animals in vivo. This phosphorylation was mediated by G protein-coupled receptor kinases (GRK) 2 and 3 and followed by rapid cointernalization of the receptor and ss-arrestin into the same endocytic vesicles. In contrast, pasireotide failed to promote substantial phosphorylation and internalization of the rat sst(2A) receptor. In the presence of octreotide or SS-14, SOM230 showed partial agonist behavior, inhibiting phosphorylation, and internalization of sst(2A). Upon overexpression of GRK2 or GRK3, pasireotide stimulated selective phosphorylation of Thr356 and Thr359 but not of Thr353 or Thr354 within the (353)TTETQRT(359) motif. Pasireotide-mediated phosphorylation led to the formation of relatively unstable beta-arrestin-sst(2A) complexes that dissociated at or near the plasma membrane. Thus, octreotide and pasireotide are equally active in inducing classical G protein-dependent signaling via the sst(2A) somatostatin receptor. Yet, we find that they promote strikingly different patterns of sst(2A) receptor phosphorylation and, hence, stimulate functionally distinct pools of beta-arrestin.

Involvement of Spinal Somatostatin Receptor SST2A in Inflammation-Induced Thermal Hyperalgesia: Ultrastructural and Behavioral Studies in Rats

Our previous results have shown that somatostatin receptor subtype SST(2A) is responsible for thermal, but not mechanical nociceptive transmission in the rat spinal cord. The present study was undertaken to further examine the ultrastructural localization of SST(2A) receptor in lamina II of the spinal dorsal horn and the role of SST(2A) receptor in thermal hyperalgesia following Complete Freund's Adjuvant (CFA)-induced inflammation. We found that SST(2A) receptors in lamina II are located primarily in postsynaptic dendrites and soma, but not in axons or synaptic terminals. CFA-induced inflammation markedly increased SST(2A) receptor-like immunoreactivity in lamina II. Paw withdrawal latency (PWL) evoked by noxious heating was obviously shortened 1 h after intraplantar injection of CFA, exhibiting thermal hyperalgesia. Pre-blocking SST(2A) activity by intrathecal pre-administration of CYN154806, a broad-spectrum antagonist of SST(2) receptor, or specific antiserum against SST(2A) receptor (anti-SST(2A)) significantly attenuated thermal hyperalgesia in a dose-dependent fashion in CFA-treated rats. But, administration of anti-SST(2A) or CYN154806 after CFA treatment had no effect upon thermal hyperalgesia. Intrathecal application of SST(2A) agonist SOM-14 at different doses prior to CFA treatment did not influence thermal hyperalgesia in inflamed rats, but at a low dose shortened PWL evoked by noxious heating in normal rats. These results suggest that spinal SST(2A) receptors play a key role in triggering the generation, but not maintenance, of thermal hyperalgesia evoked by CFA-induced inflammation. The up-regulation of SST(2A) receptors in the spinal cord may be one of the mechanisms underlying inflammation-induced thermal hyperalgesia.

Selective Loss of Somatostatin Receptor 2 in Octreotide-Resistant Growth Hormone-Secreting Adenomas

The somatostatin analog octreotide preferentially binds to somatostatin receptor (sst) 2A and to a lesser extent to sst5. Although sst2A and sst5 mRNAs are consistently expressed in GH-secreting adenomas, octreotide controls GH secretion only in 65% of acromegalic patients. Hence, we investigated the immunocytochemical expression of sst in a large group of somatotroph tumors. Acromegalic patients, cared for in a university referral center, were either operated on without pretreatment (group A, n = 14) or pretreated with octreotide [median (minimum-maximum): dose 1250 (300-1500) mug/d for 5.6 (3-9) months] before surgery (group B, n = 20). In group B octreotide reduced GH secretion by more than 50% in 14 patients (70%) (GH responders). Six patients with less than 50% GH suppression were considered GH nonresponders. We used a panel of extensively characterized antibodies to determine the immunocytochemical sst status in somatotroph adenomas and compared their expression between the groups. All group A tumors demonstrated immunoreactive sst2A, and all but one had sst5. A similar pattern was found in the GH responders of group B. In contrast, none of the GH nonresponders exhibited detectable sst2A (sst2A: GH responders vs. GH nonresponders, P < 0.0001), whereas sst5 was found in 70%. sst1 and sst3 were detected in 85 and 24% of all cases, independent of previous octreotide treatment. Our findings suggest that octreotide resistance in GH-secreting adenomas occurs due to a selective loss of sst2A. The persistent expression of sst1 and sst5 receptors suggests that these tumors are potential targets for pan-somatostatin analogs.

In vivo phosphorylation of the somatostatin 2A receptor in human tumors.

Hormone-stimulated receptor internalization and desensitization occur widely in the G protein-coupled receptor (GPCR) family. A critical first step in both these processes is thought to be receptor phosphorylation, a reaction which has been extensively characterized in cell culture. However, little is known about GPCR phosphorylation in vivo. The somatostatin (SS) receptor subtype (sst)2A is widely distributed in human neuroendocrine tumors, and SS analogs are commonly used to target this receptor for both therapy and diagnosis. In cultured pituitary cells sst2A is rapidly phosphorylated and internalized after hormone binding. The aim of the present study was to go one crucial step further and characterize the phosphorylation state of this receptor in human neuroendocrine tumors using a newly developed gel-shift assay. The receptor from a somatostatinoma was completely phosphorylated. In contrast, only unphosphorylated sst2A was present in human tumors that were not exposed to autocrine stimulation. Both in vivo and in cultured cells, the phosphorylation state of the sst2A receptor was correlated with its subcellular localization: phosphorylated receptor was mostly intracellular, whereas unphosphorylated receptor was localized at the cell surface. These results are the first to demonstrate ligand-stimulated GPCR phosphorylation in human tissue in situ, providing a crucial step toward a better understanding of receptor regulation in vivo. Analysis of sst2A phosphorylation promises to provide a sensitive indicator of the effectiveness of SS analogs in diagnostic and therapeutic situations in tumor patients.

Frequent expression of immunoreactive somatostatin receptors in cervical and endometrial cancer

Objective A variety of human cancers has been shown to express somatostatin receptors which can be utilized for in vivo tumor targeting. Although the somatostatin receptor status of mammary and ovarian cancer has been studied in detail, somatostatin receptors have not been examined in other gynecological malignancies such as cervical or endometrial carcinoma. Methods We have previously developed a panel of somatostatin receptor subtype-specific antibodies that effectively stain formalin-fixed, paraffin-embedded human tumor tissue. In the present study, we have used these antibodies to determine the somatostatin receptor status of 21 randomly selected cervical carcinomas and 28 randomly selected endometrial carcinomas. Results In cervical carcinoma, the incidence of somatostatin receptor expression was sst 2A (12/21, ∼57%) > sst 3 (9/21, ∼43%) > sst 1 (8/21, ∼38%) > sst 4 (0/21, 0%) = sst 5 (0/21, 0%). In endometrial carcinoma, the incidence of somatostatin receptor expression was sst 3 (12/28, ∼43%) > sst 2A (11/28, ∼39%) > sst 1 (9/28, ∼32%) > sst 4 (1/28, ∼4%) = sst 5 (1/28, ∼4%). Somatostatin receptor-like immunoreactivity was in most cases located at the plasma membrane and completely blocked with antigenic peptide. The expression of somatostatin receptor subtypes was independent of patient age, diagnosis, tumor stage, and histological grade. Conclusion Our findings reveal a previously unappreciated high incidence of sst 1, sst 2A, and sst 3 somatostatin receptors in human cervical and endometrial cancers and suggest that a subgroup of receptor-positive uterine carcinomas may be a potential target for diagnostic and therapeutic intervention with stable somatostatin analogs.

Somatostatin receptor immunoreactivity in the eye of the adult newt (Pleurodeles waltlii Michan)

The neuropeptide somatostatin is found in the retina of many species, yet its role in the visual process remains to be elucidated. The aim of the present study was to examine the expression and cellular localization of somatostatin receptor subtypes (sst; sst 2A, sst 2B and sst 3) in the eye of the adult newt Pleurodeles waltlii using immunohistochemistry. sst 2A immunoreactivity was observed in bipolar cells, in the inner segments of cone photoreceptors, as well as in the region corresponding to connecting cilia of rods. sst 2B immunoreactivity was not detected. sst 3 immunostaining was localized most intensely in the inner segments of cones, and in cilia of rods. These results suggest that somatostatin acting via sst 2A and sst 3 receptors may play an important role in retinal physiology of the lower vertebrates.

Differential distribution of alternatively spliced somatostatin receptor 2 isoforms (sst 2A and sst 2B) in rat spinal cord

We have recently shown that the cytoplasmic tail of the somatostatin receptor sst 2 undergoes alternative splicing giving rise to two isoforms, sst 2A and sst 2B. In the present study, we have raised polyclonal antibodies that specifically detect either sst 2A or sst 2B and used these antisera for immunocytochemical localization of the receptor proteins in the rat spinal cord. sst 2A-immunoreactivity formed a dense network consisting of neuronal perikarya and dendrites in the superficial layers of the dorsal horn. In contrast, prominent sst 2B-immunoreactivity was found on neuronal perikarya and proximal dendrites throughout the gray matter of the spinal cord. Taken together, we show that alternative carboxy-terminal splicing is involved in cell-specific expression of somatostatin receptor sst 2 isoforms in rat spinal cord, and that sst 2A and sst 2B mediate effects of somatostatin at different cellular sites.

Molecular biology of somatostatin receptor subtypes

Somatostatin (SRIF) receptors (ssts) comprise a family of heptahelical membrane proteins encoded by five related genes that map to separate chromosomes and which, with the exception of sst 1, are intronless. The ssts 1–4 display weak selectivity for SRIF-14 binding, whereas sst 5 is SRIF-28—selective. Based on structural similarity and reactivity for octapeptide and hexapeptide sst analogs, ssts 2,3 and sst 5 belong to a similar sst subclass; ssts 1–4 react poorly with these analogs and belong to a separate subclass. All five ssts are functionally coupled to inhibition of adenylyl cyclase via pertussis toxin-sensitive guanosine triphosphate (GTP)-binding proteins. mRNA for ssts 1–5 is widely expressed in brain and peripheral organs and displays an overlapping but characteristic pattern that is subtype-selective and tissue- and species-specific. All pituitary cell subsets express sst 2 and sst 5, with sst 5 being more abundant. Individual pituitary cells coexpress multiple sst subtypes. The binding pocket for SRIF-14 ligand lies deep within the membrane in transmembrane domains (TMDs) 3 to 7. Except for extracellular loop 2, it does not involve the other exofacial structures. Human (h)sst 2A and hsst 5 undergo agonist-mediated desensitization, associated with receptor internalization. The C-tail segment of hsst 5 displays positive molecular internalization signals. The ssts inhibit the growth of tumor cells directly, through blockade of mitogenic signaling leading to growth arrest and through induction of apoptosis. This process is associated with translocation of phosphotyrosine phosphatase (PTP) 1C from the cytosol to the membrane.