Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers.

The existence of receptor dimers has been proposed for several G protein-coupled receptors. However, the question of whether G protein-coupled receptor dimers are necessary for activating or modulating normal receptor function is unclear. We address this question with somatostatin receptors (SSTRs) of which there are five distinct subtypes. By using transfected mutant and wild type receptors, as well as endogenous receptors, we provide pharmacological, biochemical, and physical evidence, based on fluorescence resonance energy transfer analysis, that activation by ligand induces SSTR dimerization, both homo- and heterodimerization with other members of the SSTR family, and that dimerization alters the functional properties of the receptor such as ligand binding affinity and agonist-induced receptor internalization and up-regulation. Double label confocal fluorescence microscopy showed that when SSTR1 and SSTR5 subtypes were coexpressed in Chinese hamster ovary-K1 cells and treated with agonist they underwent internalization and were colocalized in cytoplasmic vesicles. SSTR5 formed heterodimers with SSTR1 but not with SSTR4 suggesting that heterodimerization is a specific process that is restricted to some but not all receptor subtype combinations. Direct protein interaction between different members of the SSTR subfamily defines a new level of molecular cross-talk between subtypes of the SSTR and possibly related receptor families.

Receptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity.

Somatostatin and dopamine are two major neurotransmitter systems that share a number of structural and functional characteristics. Somatostatin receptors and dopamine receptors are colocalized in neuronal subgroups, and somatostatin is involved in modulating dopamine-mediated control of motor activity. However, the molecular basis for such interaction between the two systems is unclear. Here, we show that dopamine receptor D2R and somatostatin receptor SSTR5 interact physically through hetero-oligomerization to create a novel receptor with enhanced functional activity. Our results provide evidence that receptors from different G protein (heterotrimeric guanine nucleotide binding protein)-coupled receptor families interact through oligomerization. Such direct intramembrane association defines a new level of molecular crosstalk between related G protein-coupled receptor subfamilies.

Agonist-dependent up-regulation of human somatostatin receptor type 1 requires molecular signals in the cytoplasmic C-tail.

We have previously reported that the human somatostatin receptor type 1 (hSSTR1) stably expressed in Chinese hamster ovary-K1 cells does not internalize but instead up-regulates at the membrane during continued agonist treatment (1 microM somatostatin (SST)-14 x 22 h). Here we have investigated the molecular basis of hSSTR1 up-regulation. hSSTR1 was up-regulated by SST in a time-, temperature-, and dose-dependent manner to saturable levels, in intact cells but not in membrane preparations. Although hSSTR1 was acutely desensitized to adenylyl cyclase coupling after 1 h SST-14 treatment, continued agonist exposure (22 h) restored functional effector coupling. Up-regulation was unaffected by cycloheximide but blocked by okadaic acid. Confocal fluorescence immunocytochemistry of intact and permeabilized cells showed progressive, time-dependent increase in surface hSSTR1 labeling, associated with depletion of intracellular SSTR1 immunofluorescent vesicles. To investigate the structural domains of hSSTR1 responsible for up-regulation, we constructed C-tail deletion (Delta) mutants and chimeric hSSTR1-hSSTR5 receptors. Human SSTR5 was chosen because it internalizes readily, displays potent C-tail internalization signals, and does not up-regulate. Like wild type hSSTR1, Delta C-tail hSSTR1 did not internalize and additionally lost the ability to up-regulate. Swapping the C-tail of hSSTR1 with that of hSSTR5 induced internalization (27%) but not up-regulation. Substitution of hSSTR5 C-tail with that of hSSTR1 converted the chimeric receptor to one resembling wild type hSSTR1 (poor internalization, 71% up-regulation). These results show that ligand-induced up-regulation of hSSTR1 occurs by a temperature-dependent active process of receptor recruitment from a pre-existing cytoplasmic pool to the plasma membrane. It does not require new protein synthesis or signal transduction, is sensitive to dephosphorylation events, and critically dependent on molecular signals in the receptor C-tail.

The cytoplasmic tail of the human somatostatin receptor type 5 is crucial for interaction with adenylyl cyclase and in mediating desensitization and internalization.

We have investigated the role of the cytoplasmic tail (C-tail) of the human somatostatin receptor type 5 (hSSTR5) in regulating receptor coupling to adenylyl cyclase (AC) and in mediating agonist-dependent desensitization and internalization responses. Mutant receptors with progressive C-tail truncation (Delta347, Delta338, Delta328, Delta318), Cys320 --> Ala substitution (to block palmitoylation), or Tyr304 --> Ala substitution of a putative NPXXY internalization motif were stably expressed in Chinese hamster ovary K1 cells. Except for the Tyr304 --> Ala mutant, which showed no binding, all other mutant receptors exhibited binding characteristics (Kd and Bmax) and G protein coupling comparable with wild type (wt) hSSTR5. The C-tail truncation mutants displayed progressive reduction in coupling to AC, with the Delta318 mutant showing complete loss of effector coupling. Agonist pretreatment of wt hSSTR5 led to uncoupling of AC inhibition, whereas the desensitization response of the C-tail deletion mutants was variably impaired. Compared with internalization (66% at 60 min) of wt hSSTR5, truncation of the C-tail to 318, 328, and 338 residues reduced receptor internalization to 46, 46, and 23%, respectively, whereas truncation to 347 residues slightly improved internalization (72%). Mutation of Cys320 --> Ala induced a reduction in AC coupling, desensitization, and internalization. These studies show that the C-tail of hSSTR5 serves a multifunctional role in mediating effector coupling, desensitization, and internalization. Whereas coupling to AC is dependent on the length of the C-tail, desensitization and internalization require specific structural domains. Furthermore, internalization is regulated through both positive and negative molecular signals in the C-tail and can be dissociated from the signaling and acute desensitization responses of the receptor.